Dongxia Wang

Regulation of the p300 HAT domain via a novel activation loop

The transcriptional coactivator p300 is a histone acetyltransferase (HAT) whose function is critical for regulating gene expression in mammalian cells. However, the molecular events that regulate p300 HAT activity are poorly understood. We evaluated autoacetylation of the p300 HAT protein domain to determine its function. Using expressed protein ligation, the p300 HAT protein domain was generated in hypoacetylated form and found to have reduced catalytic activity. This basal catalytic rate was stimulated by autoacetylation of several key lysine sites within an apparent activation loop motif. This post-translational modification and catalytic regulation of p300 HAT activity is conceptually analogous to the activation of most protein kinases by autophosphorylation. We therefore propose that this autoregulatory loop could influence the impact of p300 on a wide variety of signaling and transcriptional events.

Csk, a critical link of g protein signals to actin cytoskeletal reorganization.

Heterotrimeric G proteins can signal to reorganize the actin cytoskeleton, but the mechanism is unclear. Here we report that, in tyrosine kinase Csk-deficient mouse embryonic fibroblast cells, G protein (Gbetagamma, Galpha(12), Galpha(13), and Galpha(q))-induced, and G protein-coupled receptor-induced, actin stress fiber formation was completely blocked. Reintroduction of Csk into Csk-deficent cells restored the G protein-induced actin stress fiber formation. Chemical rescue experiments with catalytic mutants of Csk demonstrated that the catalytic activity of Csk was required for this process. Furthermore, we uncovered that Gbetagamma can both translocate Csk to the plasma membrane and directly increase Csk kinase activity. Our genetic and biochemical studies demonstrate that Csk plays a critical role in mediating G protein signals to actin cytoskeletal reorganization.

Substrate Conformational Restriction and CD45-catalyzed Dephosphorylation of Tail Tyrosine-phosphorylated Src Protein

Hydrolysis of the tail phosphotyrosine in Src family members is catalyzed by the protein-tyrosine phosphatase CD45, activating Src family-related signaling pathways. Using purified recombinant phospho-Src (P-Src) (amino acid residues 83–533) and purified recombinant CD45 catalytic (cytoplasmic) domain (amino acid residues 565–1268), we have analyzed the kinetic behavior of dephosphorylation. A time course of phosphatase activity showed the presence of a burst phase. By varying the concentration of P-Src, it was shown that the amplitude of this burst phase increased linearly with respect to P-Src concentration. Approximately 2% of P-Src was shown to be rapidly dephosphorylated followed by a slower linear phase. A P-Src protein substrate containing a functional point mutation in the Src homology domain 2 (SH2) led to more rapid dephosphorylation catalyzed by CD45, and this reaction showed only a single linear kinetic phase. These results were interpreted in terms of a model in which P-Src exists in a relatively slow dynamic equilibrium between “closed” and “open” conformational forms. Combined mutations in the SH2 and SH3 domain or the addition of an SH3 domain ligand peptide enhanced the accessibility of P-Src to CD45 by biasing P-Src to a more open form. Consistent with this model, a phosphotyrosine peptide that behaved as an SH2 domain binding ligand showed ∼100-fold greater affinity for unphosphorylated Srcversus P-Src. Surprisingly, P-Src possessing combined SH3 and SH2 functional inactivating point mutations was dephosphorylated by CD45 more slowly compared with P-Src completely lacking SH3 and SH2 domains. Additional data suggest that the SH3 and SH2 domains can inhibit accessibility of the P-Src tail to CD45 by interactions other than direct phosphotyrosine binding by the SH2 domain. Taken together, these results suggest how activation of Src family member signaling pathways by CD45 may be influenced by the presence or absence of ligand interactions remote from the tail.

Recommended Mass Spectrometry-Based Strategies to Identify Botulinum Neurotoxin-Containing Samples

Botulinum neurotoxins (BoNTs) cause the disease called botulism, which can be lethal. BoNTs are proteins secreted by some species of clostridia and are known to cause paralysis by interfering with nerve impulse transmission. Although the human lethal dose of BoNT is not accurately known, it is estimated to be between 0.1 μg to 70 μg, so it is important to enable detection of small amounts of these toxins. Our laboratory previously reported on the development of Endopep-MS, a mass-spectrometric‑based endopeptidase method to detect, differentiate, and quantify BoNT immunoaffinity purified from complex matrices. In this work, we describe the application of Endopep-MS for the analysis of thirteen blinded samples supplied as part of the EQuATox proficiency test. This method successfully identified the presence or absence of BoNT in all thirteen samples and was able to successfully differentiate the serotype of BoNT present in the samples, which included matrices such as buffer, milk, meat extract, and serum. Furthermore, the method yielded quantitative results which had z-scores in the range of −3 to +3 for quantification of BoNT/A containing samples. These results indicate that Endopep-MS is an excellent technique for detection, differentiation, and quantification of BoNT in complex matrices.

A Two-Stage Multiplex Method for Quantitative Analysis of Botulinum Neurotoxins Type A, B, E, and F by MALDI-TOF Mass Spectrometry

In this publication, we report on the development of a quantitative enzymatic method for the detection of four botulinum neurotoxin (BoNT) serotypes responsible for human botulism by MALDI-TOF mass spectrometry. Factors that might affect the linearity and dynamic range for detection of BoNT cleavage products were initially examined, including the amount of peptide substrate and internal standard, the timing of cleavage reaction, and the components in the reaction solution. It was found that a long incubation time produced sensitive results, but was not capable of determining higher toxin concentrations, whereas a short incubation time was less sensitive so that lower toxin concentrations were not detected. In order to overcome these limitations, a two-stage analysis strategy was applied. The first stage analysis involved a short incubation period (e.g., 30 min). If no toxin was detected at this stage, the cleavage reaction was allowed to continue and the samples were analyzed at a second time point (4 h), so that toxin levels lower than 1 mouse LD50 or 55 attomoles per milliliter (55 amol/mL) could be quantified. By combining the results from two-stage quantification, 4 or 5 orders of magnitude in dynamic range were achieved for the detection of the serotypes of BoNT/A, BoNT/B, BoNT/E, or BoNT/F. The effect of multiplexing the assay by mixing substrates for different BoNT serotypes into a single reaction was also investigated in order to reduce the numbers of the cleavage reactions and save valuable clinical samples.

Ammonia-hydrogen bromide and ammonia-hydrogen iodide complexes: Anion photoelectron and ab initio studies

The ammonia-hydrogen bromide and ammonia-hydrogen iodide, anionic heterodimers were studied by anion photoelectron spectroscopy. In complementary studies, these anions and their neutral counterparts were also investigated via ab initio theory at the coupled cluster level. In both systems, neutral NH(3)...HX dimers were predicted to be linear, hydrogen-bonded complexes, whereas their anionic dimers were found to be proton-transferred species of the form, (NH(4)(+)X(-))(-). Both experimentally measured and theoretically predicted vertical detachment energies (VDE) are in excellent agreement for both systems, with values for (NH(4)(+)Br(-))(-) being 0.65 and 0.67 eV, respectively, and values for (NH(4)(+)I(-))(-) being 0.77 and 0.81 eV, respectively. These systems are discussed in terms of our previous study of (NH(4)(+)Cl(-))(-).

Subtyping Botulinum Neurotoxins by Sequential Multiple Endoproteases In-gel Digestion Coupled with Mass Spectrometry

Botulinum neurotoxin (BoNT) is one of the most toxic substances known. BoNT is classified into seven distinct serotypes labeled A-G. Among individual serotypes, researchers have identified subtypes based on amino acid variability within a serotype and toxin variants with minor amino acid sequence differences within a subtype. BoNT subtype identification is valuable for tracing and tracking bacterial pathogens. A proteomics approach is useful for BoNT subtyping since botulism is caused by botulinum neurotoxin and does not require the presence of the bacteria or its DNA. Enzymatic digestion and peptide identification using tandem mass spectrometry determines toxin protein sequences. However, with the conventional one-step digestion method, producing sufficient numbers of detectable peptides to cover the entire protein sequence is difficult, and incomplete sequence coverage results in uncertainty in distinguishing BoNT subtypes and toxin variants because of high sequence similarity. We report here a method of multiple enzymes and sequential in-gel digestion (MESID) to characterize the BoNT protein sequence. Complementary peptide detection from toxin digestions has yielded near-complete sequence coverage for all seven BoNT serotypes. Application of the method to a BoNT-contaminated carrot juice sample resulted in the identification of 98.4% protein sequence which led to a confident determination of the toxin subtype.

Improved Sensitivity for the Qualitative and Quantitative Analysis of Active Ricin by MALDI-TOF Mass Spectrometry

Ricin is a highly toxic protein which causes cell death by blocking protein synthesis and is considered a potential bioterrorism agent. Rapid and sensitive detection of ricin toxin in various types of sample matrices is needed as an emergency requirement for public health and antibioterrorism response. An in vitro MALDI TOF MS-based activity assay that detects ricin mediated depurination of synthetic substrates was improved through optimization of the substrate, reaction conditions, and sample preparation. In this method, the ricin is captured by a specific polycolonal antibody followed by hydrolysis reaction. The ricin activity is determined by detecting the unique cleavage product of synthetic oligomer substrates. The detection of a depurinated substrate was enhanced by using a more efficient RNA substrate and optimizing buffer components, pH, and reaction temperature. In addition, the factors involved in mass spectrometry analysis, such as MALDI matrix, plate, and sample preparation, were also investigated to improve the ionization of the depurinated product and assay reproducibility. With optimized parameters, the limit of detection of 0.2 ng/mL of ricin spiked in buffer and milk was accomplished, representing more than 2 orders of magnitude enhancement in assay sensitivity. Improving assays ruggeddness or reproducibility also made it possible to quantitatively detect active ricin with 3 orders of magnitude dynamic range.