Not the usual suspect: Polymeric IgA paraprotein causes false positive results in kinetic interaction of microparticles in solution (KIMS) immunoassays.

Immunoassays are commonly used by the clinical laboratory, but paraproteins can occasionally produce erroneous results. In this study, we investigated the cause of apparent false positive results for multiple Kinetic Interaction of Microparticles in Solution (KIMS) immunoassays. Patient controls and samples containing the interference were analyzed using automated chemistry platforms, gel electrophoresis, immunofixation, affinity chromatography, and size exclusion chromatography. Our results show that IgA paraprotein caused false positive results for the KIMS measurement of three therapeutic drugs. To our knowledge, this is the first report of IgA paraprotein-causing immunoassay interference. The clinical implications of this interference are discussed.

A Single-Column Gas Chromatography Method for Quantifying Toxic Alcohols.

BACKGROUND: Rapid identification and quantification of toxic alcohols and ethylene glycol is imperative for appropriate treatment. Clinical laboratories frequently rely on direct injection gas chromatography (GC) methods, but these methods require inlet maintenance and multiple GC systems. To overcome these challenges, we developed a single-column headspace GC method for both toxic alcohols and glycols that streamlines patient sample analysis for toxic alcohol ingestion. METHODS: Optimal parameters for nonderivatized (volatile) and derivatized (glycol) plasma samples were determined using a 7890 A headspace sampler, an Agilent 7697 A GC system, a DB-200 column, and a flame ionization detector. Limit of Quantification (LoQ), linearity, imprecision, carry-over, method comparison, and interference studies were performed using quality control materials and prepared plasma samples. RESULTS: Our volatile method is linear to 3000 mg/L (ethanol) with LoQ concentrations below 20 mg/L (ethanol). The glycol method is linear to 2000 mg/L (ethylene glycol) with LoQ concentrations below 40 mg/L (ethylene glycol). Total assay impression ranged from 1.7% for ethanol to 13.3% for propylene glycol. Both methods were free of sample carryover and compared favorably with a similar clinical method at an outside laboratory. Propionic acid, an accumulating metabolite in methylmalonic acidemia that interferes with ethylene glycol identification by a different method, did not interfere with the ethylene glycol method reported here. CONCLUSIONS: Our single-column headspace GC method provides reliable, robust, and rapid identification and quantification of commonly encountered toxic alcohols. Clinical laboratories relying on direct injection Gas Chromatography (GC) for toxic alcohol analysis face challenges including frequent inlet maintenance, sample carryover, or the need for separate GC systems for volatile and glycol analysis. We summarize our development and optimization of two headspace GC methods for nonderivatized (volatile) and derivatized (glycol) plasma samples that use a single DB-200 analytical column. These methods are comparable to other GC methods, not prone to sample carryover, eliminate the need for multiple GC systems or columns, and are readily applicable to other laboratories that provide toxic alcohol analysis.

Molecular basis for receptor tyrosine kinase A-loop tyrosine transphosphorylation.

A long-standing mystery shrouds the mechanism by which catalytically repressed receptor tyrosine kinase domains accomplish transphosphorylation of activation loop (A-loop) tyrosines. Here we show that this reaction proceeds via an asymmetric complex that is thermodynamically disadvantaged because of an electrostatic repulsion between enzyme and substrate kinases. Under physiological conditions, the energetic gain resulting from ligand-induced dimerization of extracellular domains overcomes this opposing clash, stabilizing the A-loop-transphosphorylating dimer. A unique pathogenic fibroblast growth factor receptor gain-of-function mutation promotes formation of the complex responsible for phosphorylation of A-loop tyrosines by eliminating this repulsive force. We show that asymmetric complex formation induces a more phosphorylatable A-loop conformation in the substrate kinase, which in turn promotes the active state of the enzyme kinase. This explains how quantitative differences in the stability of ligand-induced extracellular dimerization promotes formation of the intracellular A-loop-transphosphorylating asymmetric complex to varying extents, thereby modulating intracellular kinase activity and signaling intensity.

Analytical and Clinical Validation of Two Commercially Available Immunoassays Used in the Detection of TSHR Antibodies.

BACKGROUND: Graves disease is caused by autoantibodies that target the thyroid-stimulating hormone receptor (TSHR). Anti-TSHR autoantibody measurement is routinely performed to differentiate between Graves disease and other causes of hyperthyroidism. We evaluated the clinical performance of a reference laboratory bioassay [the Thyretain thyroid-stimulating immunoglobulin (TSI) Bioassay by Diagnostic Hybrids] and 2 commercially available immunoassays: the TSI Bridge immunoassay by Siemens and the thyroid-stimulating hormone receptor antibody (TRAb) immunoassay by Roche. We further evaluated the analytical performance of the Siemens TSI and Roche TRAb assays. METHODS: We performed method comparisons using 125 patient specimens submitted for TSI testing for clinical purposes. Concordance of patient results was assessed between the 3 methods, and chart review was performed to further evaluate samples that generated discordant results. All 3 methods were also evaluated for potential interference caused by human chorionic gonadotropin (hCG). RESULTS: The Roche and Siemens assays demonstrated acceptable day-to-day precision, within-run precision, and precision at the clinical decision cutoffs. Despite manufacturer-defined analytical measuring ranges up to 40 IU/L, the Roche and Siemens assays were linear to 20 IU/L and 15 IU/L, respectively. hCG concentrations up to 150000 IU/L did not interfere with any of the methods evaluated. Moderate agreement between methods was observed when testing patient specimens that generated negative (≤1.3) or weakly positive (1.4-3.8) results by the Thyretain assay. One hundred percent agreement was observed when the Thyretain assay was strongly positive (≥3.9). CONCLUSIONS: The 3 commercially available anti-TSHR autoantibody measurement methods demonstrated equivalent performance in patients with untreated Graves disease. Discordant results were observed when testing specimens collected from patients undergoing treatment for Graves disease. In these patients, the Siemens TSI assay more frequently generated results consistent with clinical history, results of other laboratory tests, and imaging studies than the Thyretain Bioassay and Roche TRAb assay.

FACT Disrupts Nucleosome Structure by Binding H2A-H2B with Conserved Peptide Motifs.

FACT, a heterodimer of Spt16 and Pob3, is an essential histone chaperone. We show that the H2A-H2B binding activity that is central to FACT function resides in short acidic regions near the C termini of each subunit. Mutations throughout these regions affect binding and cause correlated phenotypes that range from mild to lethal, with the largest individual contributions unexpectedly coming from an aromatic residue and a nearby carboxylate residue within each domain. Spt16 and Pob3 bind overlapping sites on H2A-H2B, and Spt16-Pob3 heterodimers simultaneously bind two H2A-H2B dimers, the same stoichiometry as the components of a nucleosome. An Spt16:H2A-H2B crystal structure explains the biochemical and genetic data, provides a model for Pob3 binding, and implies a mechanism for FACT reorganization that we confirm biochemically. Moreover, unexpected similarity to binding of ANP32E and Swr1 with H2A.Z-H2B reveals that diverse H2A-H2B chaperones use common mechanisms of histone binding and regulating nucleosome functions.

Structure of the Spt16 middle domain reveals functional features of the histone chaperone FACT.

The histone chaperone FACT is an essential and abundant heterodimer found in all eukaryotes. Here we report a crystal structure of the middle domain of the large subunit of FACT (Spt16-M) to reveal a double pleckstrin homology architecture. This structure was found previously in the Pob3-M domain of the small subunit of FACT and in the related histone chaperone Rtt106, although Spt16-M is distinguished from these structures by the presence of an extended α-helix and a C-terminal addition. Consistent with our finding that the double pleckstrin homology structure is common to these three histone chaperones and reports that Pob3 and Rtt106 double pleckstrin homology domains bind histones H3-H4, we also find that Spt16-M binds H3-H4 with low micromolar affinity. Our structure provides a framework for interpreting a large body of genetic data regarding the physiological functions of FACT, including the identification of potential interaction surfaces for binding histones or other proteins.

To C or not to C: direct and indirect redox regulation of Src protein tyrosine kinase.

Src protein tyrosine kinase is a master regulator of cell proliferation by modulating cell metabolism, division, survival and migration, thus the mechanisms that regulate Src function are of great interest to cancer research. One emerging mode of Src regulation is its response to reactive oxygen species (ROS). ROS have historically been viewed as damaging agents in cells under oxidative stress, but recent studies establish H(2)O(2) as a secondary messenger to growth signals. A large number of cellular events respond to ROS, and many responses require the activity of Src, suggesting that Src may be a primary target of ROS. How Src kinase responds to ROS has not been established, as conflicting reports of Src activation or inactivation in response to increased concentration of ROS in the cells have been published. To determine how Src directly responds to oxidation, we investigated the effect of the redox environment on purified Src enzyme in vitro. The study reveals that Src is active in the reducing environment, and retains only 8-25% of activity in the absence of reducing agents. The inactivation is mediated by oxidation of Cys277, which leads to Src homodimers linked by a disulfide bond between the Cys277 residues of two Src monomers. A similar inactivation mechanism appears to be conserved in eight of more than 90 PTKs, including three Src family kinases and all four members of the FGFR family. The finding contradicts the view that Src is activated by oxidation, and suggests a complex response by Src to redox regulation. In this Extra View, we examine the conflicting observations in the context of complex mechanisms of Src regulation.

Direct and specific inactivation of protein tyrosine kinases in the Src and FGFR families by reversible cysteine oxidation.

Accumulating evidence suggests that protein tyrosine phosphorylation-based signaling pathways are under the regulation of reactive oxygen species. Although protein tyrosine phosphatases are directly regulated by reversible oxidation, it is not clear whether protein tyrosine kinases (PTKs) are also directly regulated by reduction/oxidation (redox). In this study we report a mechanism of direct oxidative inactivation specific for the PTKs in the Src and fibroblast growth factor receptor (FGFR) families, key enzymes in mammalian signal transduction. Src is fully active when reduced and retains 8-25% of the full activity toward various substrates when oxidized. This inactivation is caused by oxidation of a specific cysteine residue (Cys-277), which results in homodimerization of Src linked by a disulfide bridge. Cys-277 is located in the Gly loop in the catalytic domain. This cysteine residue is conserved only in 8 of the >90 PTKs in the human kinome, including 3 of the 10 Src family kinases and all 4 kinases of the FGFR family. FGFR1 is also reversibly regulated by redox because of this cysteine residue, whereas Csk, a PTK that lacks a cysteine residue at the corresponding position, is not similarly regulated. These results demonstrate a mechanism of direct redox regulation conserved in certain specific PTKs.

Bacterial expression and characterization of catalytic loop mutants of SRC protein tyrosine kinase.

Protein tyrosine kinase Src is a key enzyme in mammalian signal transduction and an important target for anticancer drug discovery. Although recombinant expression in bacterial cells offers a convenient and rapid way for producing several other protein tyrosine kinases, active Src is difficult to produce in bacterial systems. However, a kinase-defective Src mutant (due to a single point mutation, Lys295Met) is expressed strongly in bacteria. We hypothesize that the difficulty with expressing active Src in bacteria is due to toxicity caused by Src kinase activity. To test this hypothesis, we generated a series of Src mutants by altering certain residues, especially His384, in the catalytic loop and examined their expression in the bacteria and their kinase activity. The results demonstrate that Src mutants with kinase activity above a certain threshold could not be purified from a bacterial expression system, while a variety of mutants with a kinase activity below this threshold could indeed be expressed and purified. These observations support the conclusion that Src activity is toxic to the bacteria, which prevents high-level expression of fully active Src. We further demonstrated that His384, a universally conserved residue among protein tyrosine kinases, is not essential for Src catalysis or its inactivation by C-terminal tail Tyr phosphorylation. Interestingly, His384 mutants undergo autophosphorylation on Tyr416 like wild-type Src but are not activated by autophosphorylation. The potential role of His384 in Src activation by autophosphorylation is discussed in the context of Src structure.

Docking-based substrate recognition by the catalytic domain of a protein tyrosine kinase, C-terminal Src kinase (Csk).

Protein tyrosine kinases are key enzymes of mammalian signal transduction. Substrate specificity is a fundamental property that determines the specificity and fidelity of signaling by protein tyrosine kinases. However, how protein tyrosine kinases recognize the protein substrates is not well understood. C-terminal Src kinase (Csk) specifically phosphorylates Src family kinases on a C-terminal Tyr residue, which down-regulates their activities. We have previously determined that Csk recognizes Src using a substrate-docking site away from the active site. In the current study, we identified the docking determinants in Src recognized by the Csk substrate-docking site and demonstrated an interaction between the docking determinants of Src and the Csk substrate-docking site for this recognition. A similar mechanism was confirmed for Csk recognition of another Src family kinase, Yes. Although both Csk and MAP kinases used docking sites for substrate recognition, their docking sites consisted of different substructures in the catalytic domain. These results helped establish a docking-based substrate recognition mechanism for Csk. This model may provide a framework for understanding substrate recognition and specificity of other protein tyrosine kinases.