The c-Kit proto-oncogene is a receptor protein-tyrosine kinase associated with several highly malignant human cancers. Upon binding its ligand, stem cell factor (SCF), c-Kit forms an active dimer ...that autophosphorylates itself and activates a signaling cascade that induces cell growth. Disease-causing human mutations that activate SCF-independent constitutive expression of c-Kit are found in acute myelogenous leukemia, human mast cell disease, and gastrointestinal stromal tumors. We report on the phosphorylation state and crystal structure of a c-Kit product complex. The c-Kit structure is in a fully active form, with ordered kinase activation and phosphate-binding loops. These results provide key insights into the molecular basis for c-Kit kinase transactivation to assist in the design of new competitive inhibitors targeting activated mutant forms of c-Kit that are resistant to current chemotherapy regimes.
The Salmonellae PhoP-PhoQ virulence regulators induce resistance to host cationic antimicrobial peptides (CAMP) after infection of vertebrate tissues, and Mg
2+ or Ca
2+ limitation. The PhoP-PhoQ ...activated gene,
pagP, was identified as important to inducible CAMP resistance and increased acylation of lipid A, the major component of the outer leaflet of the outer membrane.
pagP mutants demonstrated increased outer membrane permeability in response to CAMP, supporting the hypothesis that increased lipid A acylation is a CAMP resistance mechanism. Similarly, in response to Mg
2+ limited growth, other enteric Gram-negative bacteria demonstrated increased lipid A acylation. Compounds that inhibit the ability to increase lipid A acylation may have utility as new antimicrobial agents.
We have developed a method to isolate and enhance the detection of phosphopeptides using liquid chromatography (LC)/mass spectrometry on a tryptic-digested protein sample. The method uses an on-line ...two-dimensional chromatography approach that consists of strong cation exchange (SCX) followed by reversed-phase (RP) chromatography with mass spectrometric detection. At pH 2.6 or lower, tryptic phosphopeptides are not retained during the first-dimension SCX chromatography step. Thus the capture of these peptides in the flow-through by the second-dimension RP trap can dramatically reduce the complexity of the phosphopeptide chromatography, resulting in little or no suppression of the signal often caused by the coeluting nonphosphorylated peptides. The method provides higher phosphopeptide recovery and less nonspecific biding of acidic peptides than the commonly used enrichment methods, such as immobilized metal affinity chromatography. Since the widely adopted multidimensional LC strategy in shotgun proteomics uses a similar SCX-RP approach, the method can be adapted to detect and characterize phosphopeptides from a complex mixture in a single experiment. Limitations of the method are also discussed.
Antimicrobial peptides are distributed throughout the animal kingdom and are a key component of innate immunity. Salmonella typhimurium regulates mechanisms of resistance to cationic antimicrobial ...peptides through the two‐component systems PhoP–PhoQ and PmrA–PmrB. Polymyxin resistance is encoded by the PmrA–PmrB regulon, whose products modify the lipopolysaccharide (LPS) core and lipid A regions with ethanolamine and add aminoarabinose to the 4′ phosphate of lipid A. Two PmrA–PmrB‐regulated S. typhimurium loci (pmrE and pmrF ) have been identified that are necessary for resistance to polymyxin and for the addition of aminoarabinose to lipid A. One locus, pmrE, contains a single gene previously identified as pagA (or ugd ) that is predicted to encode a UDP‐glucose dehydrogenase. The second locus, pmrF, is the second gene of a putative operon predicted to encode seven proteins, some with similarity to glycosyltransferases and other complex carbohydrate biosynthetic enzymes. Genes immediately flanking this putative operon are also regulated by PmrA–PmrB and/or have been associated with S. typhimurium polymyxin resistance. This work represents the first identification of non‐regulatory genes necessary for modification of lipid A and subsequent antimicrobial peptide resistance, and provides support for the hypothesis that lipid A aminoarabinose modification promotes resistance to cationic antimicrobial peptides.
A protocol is described for the production of both intracellularly expressed and secreted selenomethionyl‐derivatized recombinant proteins in baculovirus‐infected insect cells. The method results in ...the production of recombinant soluble proteins with an SeMet occupancy of ∼75% and with a recovery of ∼20% that of native protein expression. The method is independent of the percentage methionine content of the protein and is reliable and consistent. Similar results are obtained using either Spodoptera frugiperda Sf9 or Trichoplusia ni High Five insect cells as the expression host, and when cultures are grown in either shake flasks or in Wave BioReactors.
Cystic fibrosis (CF) patients develop chronic airway infections with Pseudomonas aeruginosa (PA). Pseudomonas aeruginosa synthesized lipopolysaccharide (LPS) with a variety of penta- and ...hexa-acylated lipid A structures under different environmental conditions. CF patient PA synthesized LPS with specific lipid A structures indicating unique recognition of the CF airway environment. CF-specific lipid A forms containing palmitate and aminoarabinose were associated with resistance to cationic antimicrobial peptides and increased inflammatory responses, indicating that they are likely to be involved in airway disease.
Bacterial pathogenesis requires proteins that sense host microenvironments and respond by regulating virulence gene transcription. For Salmonellae, one such regulatory system is PhoP-PhoQ, which ...regulates genes required for intracellular survival and resistance to cationic peptides. Analysis by mass spectrometry revealed that Salmonella typhimurium PhoP-PhoQ regulated structural modifications of lipid A, the host signaling portion of lipopolysaccharide (LPS), by the addition of aminoarabinose and 2-hydroxy-myristate. Structurally modified lipid A altered LPS-mediated expression of the adhesion molecule E-selectin by endothelial cells and tumor necrosis factor-α expression by adherent monocytes. Thus, altered responses to environmentally induced lipid A structural modifications may represent a mechanism for bacteria to gain advantage within host tissues.
A high-throughput online solid-phase extraction/tandem mass spectrometry (online SPE/MS/MS) system has been developed to support rapid evaluation of drug discovery compounds for possible drug-drug ...interaction (DDI). Each compound is evaluated for its DDI potential by incubating over a range of 8 test concentrations and against a panel of 6 cytochrome P450 (CYP) enzymes, 1A2, 2C8, 2C9, 2C19, 2D6, and 3A4. Previously, a postassay pooling and a 2-min gradient LC/MS/MS method had been reported to increase sample throughput, allowing for a 96-well plate of samples to be analyzed in under 4 h. The development of a new online SPE/MS/MS system has reduced the analysis time to less than 15 min per 96-well plate, translating to a 15-fold time savings compared to the 2-min LC/MS/MS method. Sampling precision without internal standard correction ranged from 3.1% to 5.6% relative standard deviation, and the carryover was determined to be between 1.0% and 4.1%. One hundred twenty in-house compounds were assayed and pooled for analyses using both the online SPE/MS/MS and LC/MS/MS, and the correlation coefficients ranged from 0.89 to 1.13, when comparing the IC50 results obtained from the 2 approaches for each of the CYP enzymes.
Summary
In this report we describe the 1500‐fold purification and characterization of the haemolytic phospholipase C (PLC) of Pseudomonas aeruginosa, the paradigm member of a novel PLC/phosphatase ...superfamily. Members include proteins from Mycobacterium tuberculosis, Bordetella spp., Francisella tularensis and Burkholderia pseudomallei. Purification involved overexpression of the plcHR1,2 operon, ion exchange chromatography and native preparative polyacrylamide gel electrophoresis. Matrix‐assisted laser desorption ionization time‐of‐flight (MALDI‐TOF) mass spectrometry confirmed the presence of two proteins in the purified sample with sizes of 17 117.2 Da (PlcR2) and 78 417 Da (PlcH). Additionally, liquid chromatography electrospray mass spectrometry (LCMS) revealed that PlcH and PlcR2 are at a stoichiometry of 1 : 1. Western blot analysis demonstrated that the enzyme purifies as a heterodimeric complex, PlcHR2. PlcHR2 is only active on choline‐containing phospholipids. It is equally active on phosphatidylcholine (PC) and sphingomyelin (SM) and is able to hydrolyse plasmenylcholine phospholipids (plasmalogens). Neither PlcHR2 nor the M. tuberculosis homologues are inhibited by D609 a widely used, competitive inhibitor of the Bacillus cereus PLC. PlcH, PlcR2, and the PlcHR2 complex bind calcium. While calcium has no detectable effect on enzymatic activity, it inhibits the haemolytic activity of PlcHR2. In addition to being required for the secretion of PlcH, the chaperone PlcR2 affects both the enzymatic and haemolytic properties of PlcH. Inclusive in these data is the con‐clusion that the members of this PC‐PLC and phosphatase family possess a novel mechanism for the recognition and hydrolysis of their respective substrates.
We describe here a high-throughput assay to support rapid evaluation of drug discovery compounds for possible drug–drug interaction (DDI). Each compound is evaluated for its DDI potential by ...incubating over a range of eight concentrations and against a panel of six cytochrome P450 (CYP) enzymes: 1A2, 2C8, 2C9, 2C19, 2D6, and 3A4. The method utilizes automated liquid handling for sample preparation, and online solid-phase extraction/tandem mass spectrometry (SPE/MS/MS) for sample analyses. The system is capable of generating two 96-well assay plates in 30 min, and completes the data acquisition and analysis of both plates in about 30 min. Many laboratories that perform the CYP inhibition screening automate only part of the processes leaving a throughput bottleneck within the workflow. The protocols described in this chapter are aimed to streamline the entire process from assay to data acquisition and processing by incorporating automation and utilizing high-precision instrument to maximize throughput and minimize bottleneck.
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