Chronic lymphocytic leukemia antibodies with a common stereotypic rearrangement recognize nonmuscle myosin heavy chain IIA.

Leukemic B lymphocytes of a large group of unrelated chronic lymphocytic leukemia (CLL) patients express an unmutated heavy chain immunoglobulin variable (V) region encoded by IGHV1-69, IGHD3-16, and IGHJ3 with nearly identical heavy and light chain complementarity-determining region 3 sequences. The likelihood that these patients developed CLL clones with identical antibody V regions randomly is highly improbable and suggests selection by a common antigen. Monoclonal antibodies (mAbs) from this stereotypic subset strongly bind cytoplasmic structures in HEp-2 cells. Therefore, HEp-2 cell extracts were immunoprecipitated with recombinant stereotypic subset-specific CLL mAbs, revealing a major protein band at approximately 225 kDa that was identified by mass spectrometry as nonmuscle myosin heavy chain IIA (MYHIIA). Reactivity of the stereotypic mAbs with MYHIIA was confirmed by Western blot and immunofluorescence colocalization with anti-MYHIIA antibody. Treatments that alter MYHIIA amounts and cytoplasmic localization resulted in a corresponding change in binding to these mAbs. The appearance of MYHIIA on the surface of cells undergoing stress or apoptosis suggests that CLL mAb may generally bind molecules exposed as a consequence of these events. Binding of CLL mAb to MYHIIA could promote the development, survival, and expansion of these leukemic cells.

Specific regulation of the adaptor protein complex AP-3 by the Arf GAP AGAP1.

Arf1 regulates membrane trafficking at several membrane sites by interacting with at least seven different vesicle coat proteins. Here, we test the hypothesis that Arf1-dependent coats are independently regulated by specific interaction with Arf GAPs. We find that the Arf GAP AGAP1 directly associates with and colocalizes with AP-3, a coat protein complex involved in trafficking in the endosomal-lysosomal system. Binding is mediated by the PH domain of AGAP1 and the delta and sigma3 subunits of AP-3. Overexpression of AGAP1 changes the cellular distribution of AP-3, and reduced expression of AGAP1 renders AP-3 resistant to brefeldin A. AGAP1 overexpression does not affect the distribution of other coat proteins, and AP-3 distribution is not affected by overexpression of other Arf GAPs. Cells overexpressing AGAP1 also exhibit increased LAMP1 trafficking via the plasma membrane. Taken together, these results support the hypothesis that AGAP1 directly and specifically regulates AP-3-dependent trafficking.

Multisite phosphorylation of adipocyte and hepatocyte phosphodiesterase 3B.

Phosphodiesterase 3B (PDE3B) is an important component of insulin and cAMP-dependent signalling pathways. In order to study phosphorylation of PDE3B, we have used an adenoviral system to express recombinant flag-tagged PDE3B in primary rat adipocytes and H4IIE hepatoma cells. Phosphorylation of PDE3B after treatment of cells with insulin, cAMP-increasing agents, or the phosphatase inhibitor, calyculin A was analyzed by two-dimensional tryptic phosphopeptide mapping and mass spectrometry. We found that PDE3B is multisite phosphorylated in adipocytes and H4IIE hepatoma cells in response to all these stimuli. Several sites were identified; serine (S)273, S296, S421, S424/5, S474 and S536 were phosphorylated in adipocyte as well as H4IIE hepatoma cells whereas S277 and S507 were phosphorylated in hepatoma cells only. Several of the sites were phosphorylated by insulin as well as cAMP-increasing hormones indicating integration of the two signalling pathways upstream of PDE3B, maybe at the level of protein kinase B.

Selective toxicity of NSC73306 in MDR1-positive cells as a new strategy to circumvent multidrug resistance in cancer.

ATP-binding cassette (ABC) proteins include the best known mediators of resistance to anticancer drugs. In particular, ABCB1 [MDR1/P-glycoprotein (P-gp)] extrudes many types of drugs from cancer cells, thereby conferring resistance to those agents. Attempts to overcome P-gp-mediated drug resistance using specific inhibitors of P-gp has had limited success and has faced many therapeutic challenges. As an alternative approach to using P-gp inhibitors, we characterize a thiosemicarbazone derivative (NSC73306) identified in a generic screen as a compound that exploits, rather than suppresses, P-gp function to induce cytotoxicity. Cytotoxic activity of NSC73306 was evaluated in vitro using human epidermoid, ovarian, and colon cancer cell lines expressing various levels of P-gp. Our findings suggest that cells become hypersensitive to NSC73306 in proportion to the increased P-gp function and multidrug resistance (MDR). Abrogation of both sensitivity to NSC73306 and resistance to P-gp substrate anticancer agents occurred with specific inhibition of P-gp function using either a P-gp inhibitor (PSC833, XR9576) or RNA interference, suggesting that cytotoxicity was linked to MDR1 function, not to other, nonspecific factors arising during the generation of resistant or transfected cells. Molecular characterization of cells selected for resistance to NSC73306 revealed loss of P-gp expression and consequent loss of the MDR phenotype. Although hypersensitivity to NSC73306 required functional expression of P-gp, biochemical assays revealed no direct interaction between NSC73306 and P-gp. This article shows that NSC73306 kills cells with intrinsic or acquired P-gp-induced MDR and indirectly acts to eliminate resistance to MDR1 substrates.

Mutation of a conserved hydrophobic patch prevents incorporation of ZP3 into the zona pellucida surrounding mouse eggs.

Three glycoproteins (ZP1, ZP2, and ZP3) are synthesized in growing mouse oocytes and secreted to form an extracellular zona pellucida that mediates sperm binding and fertilization. Each has a signal peptide to direct it into a secretory pathway, a "zona" domain implicated in matrix polymerization and a transmembrane domain from which the ectodomain must be released. Using confocal microscopy and enhanced green fluorescent protein (EGFP), the intracellular trafficking of ZP3 was observed in growing mouse oocytes. Replacement of the zona domain with EGFP did not prevent secretion of ZP3, suggesting the presence of trafficking signals and a cleavage site in the carboxyl terminus. Analysis of linker-scanning mutations of a ZP3-EGFP fusion protein in transient assays and in transgenic mice identified an eight-amino-acid hydrophobic region required for secretion and incorporation into the zona pellucida. The hydrophobic patch is conserved among mouse zona proteins and lies between a potential proprotein convertase (furin) cleavage site and the transmembrane domain. The cleavage site that releases the ectodomain from the transmembrane domain was defined by mass spectrometry of native zonae pellucidae and lies N-terminal to a proprotein convertase site that is distinct from the hydrophobic patch.

Regulation of ASAP1 by phospholipids is dependent on the interface between the PH and Arf GAP domains.

ASAP1 is an Arf GAP with a PH domain immediately N-terminal to the catalytic Arf GAP domain. PH domains are thought to regulate enzymes by binding to specific phosphoinositide lipids in membranes, thereby recruiting the enzyme to a site of action. Here, we have examined the functional relationship between the PH and Arf GAP domains. We found that GAP activity requires the cognate PH domain of ASAP1, leading us to hypothesize that the Arf GAP and PH domains directly interact to form the substrate binding site. This hypothesis was supported by the combined results of protection and hydrodynamic studies. We then examined the role of the PH domain in the regulation of Arf GAP activity. The results of saturation kinetics, limited proteolysis, FRET and fluorescence spectrometry support a model in which regulation of the GAP activity of ASAP1 involves a conformational change coincident with recruitment to a membrane surface, and a second conformational change following the specific binding of phosphatidylinositol 4,5-bisphosphate.

Attachment of neutrals during tandem mass spectrometry of sulfonic acid dyes and intermediates in an ion trap.

Several positional isomers of 2-(2-quinolinyl)-1H-indene-1,3(2H)-dione mono- and disulfonic acids prepared as reference materials for development of analytical methods involved in FDA certification of D&C Yellow No. 10 (Quinoline Yellow) were found consistently to show [MH + 14](+) ions when their electrospray- or atmospheric pressure chemical ionization-prepared MH(+) ions were subjected to collisional activation. The source of these ions was found to be the methanol used as solvent in these procedures which combined with their [MH - H(2)O](+) ions under chemical ionization conditions. The reaction was found to be sensitive to their isomeric and chemical structures and other examples of this process are reviewed.

Synthesis and structure-activity evaluation of isatin-ß-thiosemicarbazones with improved selective activity toward multidrug-resistant cells expressing P-glycoprotein.

Cancer multidrug resistance (MDR) mediated by ATP-binding cassette (ABC) transporters presents a significant unresolved clinical challenge. One strategy to resolve MDR is to develop compounds that selectively kill cells overexpressing the efflux transporter P-glycoprotein (MDR1, P-gp, ABCB1). We have previously reported structure-activity studies based around the lead compound NSC73306 (1, 1-isatin-4-(4'-methoxyphenyl)-3-thiosemicarbazone, 4.3-fold selective). Here we sought to extend this work on MDR1-selective analogues by establishing whether 1 showed "robust" activity against a range of cell lines expressing P-gp. We further aimed to synthesize and test analogues with varied substitution at the N4-position, and substitution around the N4-phenyl ring of isatin-ß-thiosemicarbazones (IBTs), to identify compounds with increased MDR1-selectivity. Compound 1 demonstrated MDR1-selectivity against all P-gp-expressing cell lines examined. This selectivity was reversed by inhibitors of P-gp ATPase activity. Structural variation at the 4'-phenyl position of 1 yielded compounds of greater MDR1-selectivity. Two of these analogues, 1-isatin-4-(4'-nitrophenyl)-3-thiosemicarbazone (22, 8.3-fold selective) and 1-isatin-4-(4'-tert-butyl phenyl)-3-thiosemicarbazone (32, 14.8-fold selective), were selected for further testing and were found to retain the activity profile of 1. These compounds are the most active IBTs identified to date.

Identification of compounds selectively killing multidrug-resistant cancer cells.

There is a great need for the development of novel chemotherapeutic agents that overcome the emergence of multidrug resistance (MDR) in cancer. We catalogued the National Cancer Institute's DTP drug repository in search of compounds showing increased toxicity in MDR cells. By comparing the sensitivity of parental cell lines with MDR derivatives, we identified 22 compounds possessing MDR-selective activity. Analysis of structural congeners led to the identification of 15 additional drugs showing increased toxicity in Pgp-expressing cells. Analysis of MDR-selective compounds led to the formulation of structure activity relationships and pharmacophore models. This data mining coupled with experimental data points to a possible mechanism of action linked to metal chelation. Taken together, the discovery of the MDR-selective compound set shows the robustness of the developing field of MDR-targeting therapy as a new strategy for resolving Pgp-mediated MDR.

Synthesis, activity, and pharmacophore development for isatin-beta-thiosemicarbazones with selective activity toward multidrug-resistant cells.

We have recently identified a new class of compounds that selectively kill cells that express P-glycoprotein (P-gp, MDR1), the ATPase efflux pump that confers multidrug resistance on cancer cells. Several isatin-beta-thiosemicarbazones from our initial study have been validated and a range of analogues synthesized and tested. A number demonstrated improved MDR1-selective activity over the lead, NSC73306 (1). Pharmacophores for cytotoxicity and MDR1 selectivity were generated to delineate the structural features required for activity. The MDR1-selective pharmacophore highlights the importance of aromatic/hydrophobic features at the N4 position of the thiosemicarbazone and the reliance on the isatin moiety as key bioisosteric contributors. Additionally, a quantitative structure-activity relationship (QSAR) model that yielded a cross-validated correlation coefficient of 0.85 effectively predicts the cytotoxicity of untested thiosemicarbazones. Together, the models serve as effective approaches for predicting structures with MDR1-selective activity and aid in directing the search for the mechanism of action of 1.

Detection of venous thromboembolism by proteomic serum biomarkers.

BACKGROUND: Available blood assays for venous thromboembolism (VTE) suffer from diminished specificity. Compared with single marker tests, such as D-dimer, a multi-marker strategy may improve diagnostic ability. We used direct mass spectrometry (MS) analysis of serum from patients with VTE to determine whether protein expression profiles would predict diagnosis. METHODS AND RESULTS: We developed a direct MS and computational approach to the proteomic analysis of serum. Using this new method, we analyzed serum from inpatients undergoing radiographic evaluation for VTE. In a balanced cohort of 76 patients, a neural network-based prediction model was built using a training subset of the cohort to first identify proteomic patterns of VTE. The proteomic patterns were then validated in a separate group of patients within the cohort. The model yielded a sensitivity of 68% and specificity of 89%, which exceeded the specificity of D-dimer assay tested by latex agglutination, ELISA, and immunoturbimetric methods (sensitivity/specificity of 63.2%/60.5%, 97.4%/21.1%, 97.4%/15.8%, respectively). We validated differences in protein expression between patients with and without VTE using more traditional gel-based analysis of the same serum samples. CONCLUSION: Protein expression analysis of serum using direct MS demonstrates potential diagnostic utility for VTE. This pilot study is the first such direct MS study to be applied to a cardiovascular disease. Differences in protein expression were identified and subsequently validated in a separate group of patients. The findings in this initial cohort can be evaluated in other independent cohorts, including patients with inflammatory conditions and chronic (but not acute) VTE, for the diagnosis of VTE.

2-Methyl-(1Z,3E)-butadiene-1,3,4-tricarboxylic acid, "isoprenetricarboxylic acid".

[reaction: see text] The synthesis of the title compound 7 from ethyl glyoxylate and dimethyl and diethyl beta-methylglutaconate is described along with its physical properties that suggest its inability to assume a cis-dienoid structure due to steric hindrance between the methyl and carboxyl groups.

Structural conservation of mouse and rat zona pellucida glycoproteins. Probing the native rat zona pellucida proteome by mass spectrometry.

The mammalian zona pellucida is an egg extracellular matrix to which sperm bind. Mouse zonae are composed of three glycoproteins (ZP1, ZP2, and ZP3), while rat zonae contain four (ZP1, ZP2, ZP3, and ZP4/ZPB). Mouse sperm bind to zonae comprised solely of mouse ZP2 and ZP3. In this report, we show that rat sperm also bind to these zonae, indicating that ZP2 and ZP3 contain a "minimum structure(s)" to which rodent sperm can bind, and ZP1 and ZP4/ZPB are dispensable in these two rodents. These data are consistent with our mass spectrometric analysis of the native rat zona pellucida proteome (defined as the fraction of the total rat proteome to which the zonae glycoproteins contribute) demonstrating that the rat zonae glycoproteins share a high degree of conservation of structural features with respect to their mouse counterparts. The primary sequences of the rat zonae proteins have been deduced from cDNA. Each zona protein undergoes extensive co- and post-translational modification prior to its secretion and incorporation into an extracellular zona matrix. Each has a predicted N-terminal signal peptide that is cleaved off once protein translation begins and an anchoring C-terminal transmembrane domain from which the mature protein is released. Mass spectrometric analysis with a limited amount of native material allowed determination of the mature N-termini of rat ZP1 and ZP3, both of which are characterized by cyclization of glutamine to pyroglutamate; the N-terminus of ZP2 was identified by Edman degradation. The mature C-termini of ZP1 and ZP3 end two amino acids upstream of a conserved dibasic residue that is part of, but distinct from, the consensus furin cleavage sequence, while the C-terminus of ZP2 was not determined. Each zona protein contains a "zona domain" with eight conserved cysteine residues that is thought to play a role in the polymerization of the zona proteins into matrix filaments. Partial disulfide bond assignment indicates that the intramolecular disulfide patterns in rat ZP1, ZP2, and ZP3 are identical to those of their corresponding mouse counterparts. Last, nearly all potential N-glycosylation sites are occupied in the rat zonae glycoproteins (three of three for ZP1, six or seven of seven for ZP2, and four or five of six for ZP3). In comparison, potential O-glycosylation sites are numerous (59-83 Ser/Thr residues), but only two regions were observed to carry O-glycans in rat ZP3.

Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence.

Here we report the structural characterization of the product formed from the reaction between hydroethidine (HE) and superoxide (O(2)(.-)). By using mass spectral and NMR techniques, the chemical structure of this product was determined as 2-hydroxyethidium (2-OH-E(+)). By using an authentic standard, we developed an HPLC approach to detect and quantitate the reaction product of HE and O(2)(.-) formed in bovine aortic endothelial cells after treatment with menadione or antimycin A to induce intracellular reactive oxygen species. Concomitantly, we used a spin trap, 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO), to detect and identify the structure of reactive oxygen species formed. BMPO trapped the O(2)(.-) that formed extracellularly and was detected as the BMPO-OH adduct during use of the EPR technique. BMPO, being cell-permeable, inhibited the intracellular formation of 2-OH-E(+). However, the intracellular BMPO spin adduct was not detected. The definitive characterization of the reaction product of O(2)(.-) with HE described here forms the basis of an unambiguous assay for intracellular detection and quantitation of O(2)(.-). Analysis of the fluorescence characteristics of ethidium (E(+)) and 2-OH-E(+) strongly suggests that the currently available fluorescence methodology is not suitable for quantitating intracellular O(2)(.-). We conclude that the HPLC/fluorescence assay using HE as a probe is more suitable [corrected] for detecting intracellular O(2)(.-).

Identification of carboxypeptidase N as an enzyme responsible for C-terminal cleavage of stromal cell-derived factor-1alpha in the circulation.

The chemokine stromal-derived factor-1alpha (SDF-1alpha) is an essential regulator of hematopoiesis, lymphocyte homing, pre-B-cell growth, and angiogenesis. As SDF-1alpha is constitutively expressed in many tissues, chemokine function is mostly regulated by proteolytic degradation. Human serum cleaves the 68-amino acid chemokine, SDF-1alpha, at both termini. The enzyme or enzymes responsible for the removal of the carboxy-terminal lysine from SDF-1alpha, leading to significant reduction in biologic activity, have not been identified. Using a new biochemical assay for measuring the carboxy-terminal cleavage activity, we purified from serum and plasma a peptidase that specifically removes the carboxy-terminal lysine from SDF-1alpha and identified it as carboxypeptidase N (CPN, also known as kininase I, arginine carboxypeptidase, and anaphylotoxin inactivator). We demonstrate that SDF-1alpha in serum and plasma lacks the carboxy terminal lysine, and depletion of CPN from serum and plasma significantly reduces the SDF-1alpha carboxypeptidase activity. Purified CPN effectively and specifically removes the carboxy-terminal lysine from SDF-1alpha and significantly reduces the chemokine's biologic activity as a pre-B-cell growth factor and chemoattractant. Thus, in addition to its role as a regulator of the biologic activity of kinins and anaphylatoxins, CPN is an important regulator of the biologic activity of SDF-1alpha by reducing the chemokine-specific activity.

Intramolecular cross-linking experiments on cytochrome c and ribonuclease A using an isotope multiplet method.

Mass spectral analysis of tryptic digests of cross-linked proteins offers considerable promise as a simple technique to probe protein structure and study protein-protein interactions. We describe the use of a 1:1 mixture of isotopically labeled and unlabeled cross-linkers, disuccinimidyladipate (DSA) and dimethyladipimidate (DMA), to enhance visualization of cross-linked peptides in a tryptic digest. Optimized intramolecular reactions of cytochrome c and ribonuclease A (RNase A) with DSA yielded an average of two cross-links per protein molecule. After digestion of the cross-linked cytochrome c with trypsin and analysis by liquid chromatography/mass spectrometry (LC/MS) and matrix-assisted laser desorption/ionization (MALDI), eight modified peptides, five cross-linked and two end-capped, were detected by virtue of their doublet character. An eighth modified peptide's identity remained ambiguous because of its inability to fragment. The lysine-lysine distance constraints obtained are discussed in the context of the known NMR and X-ray structures of cytochrome c. Analysis of cross-linked RNase A by LC/MS and MALDI yielded nine modified peptides, four of which were modified twice, as indicated by the isotopic triplets. Although seven of these peptides contained cross-links, few distance constraints were gained due to the fact that the cross-linked products were variations of modification of the same three lysine residues.

Divinyl sulfone as a postdigestion modifier for enhancing the a(1) Ion in MS/MS and postsource decay: potential applications in proteomics.

Divinyl sulfone reacts at pH 8-9 with the alpha-amino groups of N-terminal residues, proline, the epsilon-amino groups of lysine, and the histidine side chains of peptides. This reaction leads to great enhancement of the abundance of the normally weak or missing "a(1)" fragment ion in MS/MS analysis defining the N-terminal residue of a peptide in a digest. This provides "one-step Edman-like" information that, together with a fairly accurately determined mass, often enables one to correctly identify a protein or family of proteins. The applicability of this procedure in proteomics was demonstrated with several peptides and tryptic digests of protein mixtures by LC-MS/MS experiments using a QTOF and MALDI-PSD analyses. Advantages of this approach are its simple chemistry, retention of charge multiplicity, and possibly, shortening of database search time. Used with other MS/MS data, it provides higher confidence in the scores and identification of a protein found in peptide mass fingerprinting. Moreover, this approach has an advantage in "de novo" sequencing due to its ability to decipher the first amino acid of a peptide whose information is normally unavailable in MS/MS spectra.

Structural characterization of native mouse zona pellucida proteins using mass spectrometry.

The zona pellucida is an extracellular matrix consisting of three glycoproteins that surrounds mammalian eggs and mediates fertilization. The primary structures of mouse ZP1, ZP2, and ZP3 have been deduced from cDNA. Each has a predicted signal peptide and a transmembrane domain from which an ectodomain must be released. All three zona proteins undergo extensive co- and post-translational modifications important for secretion and assembly of the zona matrix. In this report, native zonae pellucidae were isolated and structural features of individual zona proteins within the mixture were determined by high resolution electrospray mass spectrometry. Complete coverage of the primary structure of native ZP3, 96% of ZP2, and 56% of ZP1, the least abundant zona protein, was obtained. Partial disulfide bond assignments were made for each zona protein, and the size of the processed, native protein was determined. The N termini of ZP1 and ZP3, but not ZP2, were blocked by cyclization of glutamine to pyroglutamate. The C termini of ZP1, ZP2, and ZP3 lie upstream of a dibasic motif, which is part of, but distinct from, a proprotein convertase cleavage site. The zona proteins are highly glycosylated and 4/4 potential N-linkage sites on ZP1, 6/6 on ZP2, and 5/6 on ZP3 are occupied. Potential O-linked carbohydrate sites are more ubiquitous, but less utilized.