Martin Sadilek

Interfacial Kinetic and Binding Properties of the Complete Set of Human and Mouse Groups I, II, V, X, and XII Secreted Phospholipases A2

Expression of the full set of human and mouse groups I, II, V, X, and XII secreted phospholipases A2 (sPLA2s) in Escherichia coli and insect cells has provided pure recombinant enzymes for detailed comparative interfacial kinetic and binding studies. The set of mammalian sPLA2s display dramatically different sensitivity to dithiothreitol. The specific activity for the hydrolysis of vesicles of differing phospholipid composition by these enzymes varies by up to 4 orders of magnitude, and yet all enzymes display similar catalytic site specificity toward phospholipids with different polar head groups. Discrimination between sn-2 polyunsaturated versus saturated fatty acyl chains is <6-fold. These enzymes display apparent dissociation constants for activation by calcium in the 1–225 μm range, depending on the phospholipid substrate. Analysis of the inhibition by a set of 12 active site-directed, competitive inhibitors reveals a large variation in the potency among the mammalian sPLA2s, with Me-Indoxam being the most generally potent sPLA2 inhibitor. A dramatic correlation exists between the ability of the sPLA2s to hydrolyze phosphatidylcholine-rich vesicles efficiently in vitro and the ability to release arachidonic acid when added exogenously to mammalian cells; the group V and X sPLA2s are uniquely efficient in this regard.

Molecular Mechanism of Calcium Channel Regulation in the Fight-or-Flight Response

Stimulation of cardiac Ca2+ channel activity by PKA in the fight-or-flight response requires an autoinhibitory complex with the distal C-terminal domain and a kinase anchoring protein. The Complex Regulation of Heartbeat The pounding heart associated with moments of intense exercise, stress, or terror depends on activation of the sympathetic nervous system and the consequent increase in calcium influx through cardiac calcium channels. This increase in calcium influx is mediated through stimulation of protein kinase A (PKA) by the β-adrenergic signaling pathway and the ensuing PKA-dependent phosphorylation of the CaV1.2 channel. Fuller et al. reconstituted the CaV1.2 channel signaling complex in non-muscle cells and discovered that PKA-dependent stimulation of channel activity required both the proper ratio of CaV1.2 to the scaffolding protein A-kinase–anchoring protein 15 (AKAP15) and noncovalent association with the distal C-terminal domain of CaV1.2, which is proteolytically processed in vivo. The autoinhibitory effect of noncovalent association of the distal C-terminal domain is relieved by phosphorylation of a serine residue at the interface between the distal and the proximal C-terminal domains, leading to disinhibition of channel activity in the fight-or-flight response. During the fight-or-flight response, the sympathetic nervous system stimulates L-type calcium ion (Ca2+) currents conducted by CaV1 channels through activation of β-adrenergic receptors, adenylyl cyclase, and phosphorylation by adenosine 3′,5′-monophosphate–dependent protein kinase [also known as protein kinase A (PKA)], increasing contractility of skeletal and cardiac muscles. We reconstituted this regulation of cardiac CaV1.2 channels in non-muscle cells by forming an autoinhibitory signaling complex composed of CaV1.2Δ1800 (a form of the channel truncated at the in vivo site of proteolytic processing), its noncovalently associated distal carboxyl-terminal domain, the auxiliary α2δ1 and β2b subunits, and A-kinase anchoring protein 15 (AKAP15). A factor of 3.6 range of CaV1.2 channel activity was observed from a minimum in the presence of protein kinase inhibitors to a maximum upon activation of adenylyl cyclase. Basal CaV1.2 channel activity in unstimulated cells was regulated by phosphorylation of serine-1700 and threonine-1704, two residues located at the interface between the distal and the proximal carboxyl-terminal regulatory domains, whereas further stimulation of channel activity through the PKA signaling pathway only required phosphorylation of serine-1700. Our results define a conceptual framework for CaV1.2 channel regulation and identify sites of phosphorylation that regulate channel activity.

Simplified capillary electrophoresis nanospray sheath‐flow interface for high efficiency and sensitive peptide analysis

We report a simple nanospray sheath-flow interface for capillary electrophoresis. This interface relies on electrokinetic flow to drive both the separation and the electrospray; no mechanical pump is used for the sheath flow. This system was interfaced with an LCQ mass spectrometer. The best results were observed with a 2-microm diameter emitter tip and a 1-mm spacing between the separation capillary tip and the emitter tip. Under these conditions, mass detection limits (3sigma) of 100 amol were obtained for insulin receptor fragment 1142-1153. The separation efficiency exceeded 200,000 plates for this compound. The relative standard deviation generated during continual infusion of a 50 microM solution of angiotensin II was 2% for the total ion count and 3% for the extracted ion count over a 40-min period. Finally, the interface was also demonstrated for negative ion mode.

c-Myc activates multiple metabolic networks to generate substrates for cell-cycle entry.

Cell proliferation requires the coordinated activity of cytosolic and mitochondrial metabolic pathways to provide ATP and building blocks for DNA, RNA and protein synthesis. Many metabolic pathway genes are targets of the c-myc oncogene and cell-cycle regulator. However, the contribution of c-Myc to the activation of cytosolic and mitochondrial metabolic networks during cell-cycle entry is unknown. Here, we report the metabolic fates of [U-13C] glucose in serum-stimulated myc−/− and myc+/+ fibroblasts by 13C isotopomer NMR analysis. We demonstrate that endogenous c-myc increased 13C labeling of ribose sugars, purines and amino acids, indicating partitioning of glucose carbons into C1/folate and pentose phosphate pathways, and increased tricarboxylic acid cycle turnover at the expense of anaplerotic flux. Myc expression also increased global O-linked N-acetylglucosamine protein modification, and inhibition of hexosamine biosynthesis selectively reduced growth of Myc-expressing cells, suggesting its importance in Myc-induced proliferation. These data reveal a central organizing function for the Myc oncogene in the metabolism of cycling cells. The pervasive deregulation of this oncogene in human cancers may be explained by its function in directing metabolic networks required for cell proliferation.

Tandem Mass Spectrometry for the Direct Assay of Lysosomal Enzymes in Dried Blood Spots:Application to Screening Newborns for Mucopolysaccharidosis IVA

BACKGROUND Treatments are being developed for an increasing number of mucopolysaccharidoses, and early diagnosis is expected to be necessary to maximize the benefits of therapy. Therefore, we developed an assay for N-acetylgalactosamine-6-sulfate sulfatase (GALNS), the enzyme deficient in mucopolysaccharidosis IVA (Morquio A syndrome), that is applicable for clinical diagnosis. METHODS A novel substrate for GALNS was synthesized for a new enzyme activity assay that is based on tandem mass spectrometry and uses dried blood spots (DBSs) as the enzyme source. We optimized the assay conditions, including the substrate concentration, reaction pH, lead formate concentration, incubation time, punch size of the DBS, and mass spectrometer conditions. We also assessed inter- and intraassay variation. RESULTS The assay uses either solid-phase or liquid-phase extraction before analysis by mass spectrometry. An evaluation of blood spots from 90 randomly chosen healthy newborns and 9 patients with Morquio A syndrome showed a well-defined interval between their respective enzyme activities. Inter- and intraassay imprecision was <10%. CONCLUSIONS This tandem mass spectrometry assay requires a minimal number of sample-preparation steps, thus making it easy to implement. The assay has the potential to be adopted for early diagnosis of Morquio A syndrome. We believe this assay could be performed in a multiplex fashion with assays for other lysosomal enzymes.

Improved sensitivity mass spectrometric detection of eicosanoids by charge reversal derivatization.

Combined liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) is a powerful method for the analysis of oxygenated metabolites of polyunsaturated fatty acids including eicosanoids. Here we describe the synthesis of a new derivatization reagent N-(4-aminomethylphenyl)pyridinium (AMPP) that can be coupled to eicosanoids via an amide linkage in quantitative yield. Conversion of the carboxylic acid of eicosanoids to a cationic AMPP amide improves sensitivity of detection by 10- to 20-fold compared to negative mode electrospray ionization detection of underivatized analytes. This charge reversal derivatization allows detection of cations rather than anions in the electrospray ionization mass spectrometer, which enhances sensitivity. Another factor is that AMPP amides undergo considerable collision-induced dissociation in the analyte portion rather than exclusively in the cationic tag portion, which allows isobaric derivatives to be distinguished by tandem mass spectrometry, and this further enhances sensitivity and specificity. This simple derivatization method allows prostaglandins, thromboxane B(2), leukotriene B(4), hydroxyeicosatetraenoic acid isomers, and arachidonic acid to be quantified in complex biological samples with limits of quantification in the 200-900 fg range. One can anticipate that the AMPP derivatization method can be extended to other carboxylic acid analytes for enhanced sensitivity detection.

Tryptic Peptide Analysis of Ceruloplasmin in Dried Blood Spots Using Liquid Chromatography–Tandem Mass Spectrometry: Application to Newborn Screening

BACKGROUND Newborn screening to identify infants with treatable congenital disorders is carried out worldwide. Recent tandem mass spectrometry (MS/MS) applications have markedly expanded the ability to screen for >50 metabolic diseases with a single dried blood spot (DBS). The feature that makes metabolic disorders particularly amenable to screening is the presence of specific small-molecule metabolites. Many treatable disorders such as Wilson disease, however, are characterized by absent or diminished large proteins in plasma or within circulating blood cells, for which there are currently no cost-effective screening methods. METHODS We developed an assay for quantifying ceruloplasmin (CP) in DBS for newborn screening of Wilson disease. CP-specific peptides from DBS samples digested by trypsin were quantified using isotopically labeled peptide internal standards and liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS). RESULTS The calibration curve was linear from 20 to 95 mg/dL (200-950 mg/L). Intraassay imprecision (mean CV) for CP concentrations of 25, 35, and 55 mg/dL (250, 350, and 550 mg/L) was 9.2%, 10.7%, and 10.2%, respectively. Interassay imprecision for 19 different batches was 8.9%, 5.8%, and 6.9%. A method comparison study on previously tested patient samples for CP gave comparable results with lower limit of quantification, around 0.7 mg/dL (7 mg/L). CONCLUSIONS Our study supports that newborn screening for Wilson disease is feasible using LC-MS/MS assay for CP quantification in DBS after tryptic digestion. This approach should be applicable to newborn screening for other treatable genetic conditions, such as primary immunodeficiencies, that have large proteins as biomarkers.

Hydrolysis products of cAMP analogs cause transformation of Trypanosoma brucei from slender to stumpy-like forms.

African sleeping sickness is a disease caused by Trypanosoma brucei. T. brucei proliferate rapidly in the mammalian bloodstream as long, slender forms, but at higher population densities they transform into nondividing, short, stumpy forms. This is thought to be a mechanism adopted by T. brucei to establish a stable host–parasite relationship and to allow a transition into the insect stage of its life cycle. Earlier studies have suggested a role for cAMP in mediating this transformation. In this study, using membrane-permeable nucleotide analogs, we show that it is not the cAMP analogs themselves but rather the hydrolyzed products of membrane-permeable cAMP analogs that prevent proliferation of T. brucei. The metabolic products are more potent than the cAMP analogs, and hydrolysis-resistant cAMP analogs are not antiproliferative. We further show that the antiproliferative effect of these membrane-permeable adenosine analogs is caused by transformation into forms resembling short, stumpy bloodstream forms. These data suggest that the slender-to-stumpy transformation of T. brucei may not be mediated directly by cAMP and also raise the possibility of using such adenosine analogs as antitrypanosomal drugs.

Tandem Mass Spectrometry for the Direct Assay of Lysosomal Enzymes in Dried Blood Spots: Application to Screening Newborns for Mucopolysaccharidosis II (Hunter Syndrome)

We have developed a tandem mass spectrometry based assay of iduronate-2-sulfatase (IdS) activity for the neonatal detection of mucopolysaccharidosis II (MPS-II, Hunter Syndrome). The assay uses a newly designed synthetic substrate (IdS-S) consisting of α-L-iduronate-2-sulfate, which is glycosidically conjugated to a coumarin and a linker containing a tert-butyloxycarbamido group. A short synthesis of the substrate has been developed that has the potential of being scaled to multigram quantities. Sulfate hydrolysis of IdS-S by IdS found within a 3 mm dried blood spot specifically produces a nonsulfated product (IdS-P) which is detected by electrospray tandem mass spectrometry and quantified using a deuterium-labeled internal standard, both carried out in positive ion mode. Analysis of DBS from 75 random human newborns showed IdS activities in the range of 4.8-16.2 (mean 9.1) μmol/(h L of blood), which were clearly distinguished from the activities measured for 14 MPS-II patients at 0.17-0.52 (mean 0.29) μmol/(h L of blood). The assay shows low blank activity, 0.15 ± 0.03 μmol/(h L of blood). The within-assay coefficient of variation (CV) was 3.1% while the interassay CV was 15%.

A class of sterol 14-demethylase inhibitors as anti-Trypanosoma cruzi agents

Chronic infection with the protozoan parasite Trypanosoma cruzi is a major cause of morbidity and mortality in Latin America. Drug treatments for the associated illness, Chagas disease, are toxic and frequently unsuccessful. In a screening effort against the drug target protein farnesyltransferase, we identified a series of disubstituted imidazoles with highly potent anti-T. cruzi activity that apparently acted through a mechanism independent of protein farnesylation. Metabolic labeling studies of T. cruzi suggested that sterol biosynthesis was inhibited. Combined GC/MS analysis confirmed depletion of cellular sterols and suggested that the site of action was sterol 14-demethylase, a cytochrome P450 enzyme. Spectral studies with recombinant T. cruzi sterol 14-demethylase demonstrated that the compounds bind directly to this enzyme. Two of the compounds were well absorbed when given orally to mice, gave sustained plasma levels, and were well tolerated. The compounds were administered orally to mice with acute T. cruzi infection and caused dramatic decrease in parasitemia and led to 100% survival. These disubstituted imidazole compounds can be prepared by a relatively short synthetic route and represent a structural class with potent anti-T. cruzi activity.