Synthesizing Signaling Pathways from Temporal Phosphoproteomic Data.

We present a method for automatically discovering signaling pathways from time-resolved phosphoproteomic data. The Temporal Pathway Synthesizer (TPS) algorithm uses constraint-solving techniques first developed in the context of formal verification to explore paths in an interaction network. It systematically eliminates all candidate structures for a signaling pathway where a protein is activated or inactivated before its upstream regulators. The algorithm can model more than one hundred thousand dynamic phosphosites and can discover pathway members that are not differentially phosphorylated. By analyzing temporal data, TPS defines signaling cascades without needing to experimentally perturb individual proteins. It recovers known pathways and proposes pathway connections when applied to the human epidermal growth factor and yeast osmotic stress responses. Independent kinase mutant studies validate predicted substrates in the TPS osmotic stress pathway.

Development of Selected Reaction Monitoring Methods to Systematically Quantify Kinase Abundance and Phosphorylation Stoichiometry in Human Samples.

Protein phosphorylation, one of the most common types of post-translational modifications, is the central regulatory mechanism of cellular signaling networks. In human cells, thousands of proteins are continuously and dynamically phosphorylated and dephosphorylated at specific sites and times in response to external and internal stimuli. Reversible phosphorylation is facilitated by the action of two protein superfamilies: kinases and phosphatases. Kinases play an essential role in almost every relevant physiological process in human cells and their deregulation is linked to pathologies ranging from cancer to autoimmune diseases.Systematic identification of kinases expressed in a particular cell type, quantification of their abundance, and precise determination of their phosphorylation stoichiometry are essential to understand the cellular signaling networks and physiology of a sample. Our protocol outlines the steps to build and use a high-throughput, comprehensive, modular, and robust selected reaction monitoring (SRM) proteomics framework to facilitate quantification of the kinome state in research or clinical human samples.

Pediatric Intensive Care Unit rounding improvements.

Medical rounds in the Pediatric Intensive Care Unit (PICU) took about four hours each day to complete. The length of rounds was affecting the advancement of care of the patients, the engagement of sub-specialty providers who needed to be present on other rounds, and the engagement of the PICU faculty and staff due to overburdening waste created by the long duration of rounds. Specific interventions were identified aimed at reducing the duration of rounds each day, increasing engagement of the rounding team and satisfying the needs of the patients and families. Post-improvement results were that rounding times were reduced to two hours each day and were pre-scheduled for families, the burden of excess work was lifted for attending physicians, and the presentation expectations during rounds were specified by role. Quality and safety were improved through standard work and auditing.

Trypanosoma brucei Tb927.2.6100 is an essential protein associated with kinetoplast DNA.

The mitochondrial DNA of trypanosomatid protozoa consists of a complex, intercatenated network of tens of maxicircles and thousands of minicircles. This structure, called kinetoplast DNA (kDNA), requires numerous proteins and multiprotein complexes for replication, segregation, and transcription. In this study, we used a proteomic approach to identify proteins that are associated with the kDNA network. We identified a novel protein encoded by Tb927.2.6100 that was present in a fraction enriched for kDNA and colocalized the protein with kDNA by fluorescence microscopy. RNA interference (RNAi) knockdown of its expression resulted in a growth defect and changes in the proportion of kinetoplasts and nuclei in the cell population. RNAi also resulted in shrinkage and loss of the kinetoplasts, loss of maxicircle and minicircle components of kDNA at similar rates, and (perhaps secondarily) loss of edited and pre-edited mRNA. These results indicate that the Tb927.2.6100 protein is essential for the maintenance of kDNA.

The archaeo-eukaryotic primase of plasmid pRN1 requires a helix bundle domain for faithful primer synthesis.

The plasmid pRN1 encodes for a multifunctional replication protein with primase, DNA polymerase and helicase activity. The minimal region required for primase activity encompasses amino-acid residues 40-370. While the N-terminal part of that minimal region (residues 47-247) folds into the prim/pol domain and bears the active site, the structure and function of the C-terminal part (residues 248-370) is unknown. Here we show that the C-terminal part of the minimal region folds into a compact domain with six helices and is stabilized by a disulfide bond. Three helices superimpose well with the C-terminal domain of the primase of the bacterial broad host range plasmid RSF1010. Structure-based site-directed mutagenesis shows that the C-terminal helix of the helix bundle domain is required for primase activity although it is distant to the active site in the crystallized conformation. Furthermore, we identified mutants of the C-terminal domain, which are defective in template binding, dinucleotide formation and conformation change prior to DNA extension.

Factors controlling the carbon isotope fractionation of tetra- and trichloroethene during reductive dechlorination by Sulfurospirillum ssp. and Desulfitobacterium sp. strain PCE-S.

Carbon stable isotope fractionation of tetrachloroethene (PCE) and trichloroethene (TCE) was investigated during reductive dechlorination. Growing cells of Sulfurospirillum multivorans, Sulfurospirillum halorespirans, or Desulfitobacterium sp. strain PCE-S, the respective crude extracts and the abiotic reaction with cyanocobalamin (vitamin B(12)) were used. Fractionation of TCE (alphaC=1.0132-1.0187) by S. multivorans was more than one order of magnitude higher than values previously observed for tetrachloroethene (PCE) (alphaC=1.00042-1.0017). Similar differences in fractionation were observed during reductive dehalogenation by the close relative S. halorespirans with alphaC=1.0046-1.032 and alphaC=1.0187-1.0229 for PCE and TCE respectively. TCE carbon isotope fractionation (alphaC=1.0150) by the purified PCE-reductive dehalogenase from S. multivorans was more than one order of magnitude higher than fractionation of PCE (alphaC=1.0017). Carbon isotope fractionation of TCE by Desulfitobacterium sp. strain PCE-S (alphaC=1.0109-1.0122) as well as during the abiotic reaction with cyanocobalamin (alphaC=1.0154) was in a similar range to previously reported values for fractionation by mixed microbial cultures. In contrast with previous results with PCE, no effects due to rate limitations, uptake or transport of the substrate to the reactive site could be observed during TCE dechlorination. Our results show that prior to a mechanistic interpretation of stable isotope fractionation factors it has to be carefully verified how other factors such as uptake or transport affect the isotope fractionation during degradation experiments with microbial cultures.

Properties of an unusual DNA primase from an archaeal plasmid.

Primases are specialized DNA-dependent RNA polymerases that synthesize a short oligoribonucleotide complementary to single-stranded template DNA. In the context of cellular DNA replication, primases are indispensable since DNA polymerases are not able to start DNA polymerization de novo. The primase activity of the replication protein from the archaeal plasmid pRN1 synthesizes a rather unusual mixed primer consisting of a single ribonucleotide at the 5' end followed by seven deoxynucleotides. Ribonucleotides and deoxynucleotides are strictly required at the respective positions within the primer. Furthermore, in contrast to other archaeo-eukaryotic primases, the primase activity is highly sequence-specific and requires the trinucleotide motif GTG in the template. Primer synthesis starts outside of the recognition motif, immediately 5' to the recognition motif. The fidelity of the primase synthesis is high, as non-complementary bases are not incorporated into the primer.

Stable isotope fractionation of tetrachloroethene during reductive dechlorination by Sulfurospirillum multivorans and Desulfitobacterium sp. strain PCE-S and abiotic reactions with cyanocobalamin.

Carbon stable isotope fractionation of tetrachloroethene (PCE) during reductive dechlorination by whole cells and crude extracts of Sulfurospirillum multivorans and Desulfitobacterium sp. strain PCE-S and the abiotic reaction with cyanocobalamin (vitamin B12) was studied. Fractionation was largest during the reaction with cyanocobalamin with alphaC = 1.0132. Stable isotope fractionation was lower but still in a similar order of magnitude for Desulfitobacterium sp. PCE-S (alphaC = 1.0052 to 1.0098). The isotope fractionation of PCE during dehalogenation by S. multivorans was lower by 1 order of magnitude (alphaC = 1.00042 to 1.0017). Additionally, an increase in isotope fractionation was observed with a decrease in cell integrity for both strains. For Desulfitobacterium sp. strain PCE-S, the carbon stable isotope fractionation factors were 1.0052 and 1.0089 for growing cells and crude extracts, respectively. For S. multivorans, alphaC values were 1.00042, 1.00097, and 1.0017 for growing cells, crude extracts, and the purified PCE reductive dehalogenase, respectively. For the field application of stable isotope fractionation, care is needed as fractionation may vary by more than an order of magnitude depending on the bacteria present, responsible for degradation.

Synthesis and characterization of metal binding pseudotripeptides.

Metal complexes with peptide or pseudopeptide type ligands can serve as good model compounds for a deeper understanding of enzymatic catalysis, but ligands with a high selectivity for different transition metal cations are hard to find due to the rather flexible nature of peptides. Since such ligands would be the sine qua non condition for the synthesis of heterodinuclear peptide metal complexes with catalytic activity, the search for small, affine and selective metal chelating sequences is of interest. Using four different amino acids (His, Lys, Asp, Glu) a set of 16 pseudotripeptides of the common structure Bz-AS1-Sar-AS2-NH2 has been synthesized, purified and characterized by mass spectrometry and 1H-NMR. Their ability to form metal complexes has been investigated leading to short motifs capable of selectively binding only one or two transition metal cations with high affinity. As expected, the complexation of transition metal cations by pseudotripeptides is strongly dependent not only on the amino acid composition, but also on the sequence with regard to the stability of the resulting complexes, as well as the selectivity of the ligands towards Cu2+, Co2+, Ni2+, Zn2+ and Mn2+.