Search

Guanabenz, a clinically used anti-hypertensive agent, inhibits the P450-like enzyme neuronal NO-synthase (nNOS) and enhances its ubiquitination and degradation. To better understand the molecular trigger for nNOS ubiquitination and degradation, we characterized the mechanism of guanabenz inhibition of nNOS and identified the site of ubiquitin attachment to the enzyme. Using purified nNOS and an in vitro system, we found that guanabenz treatment leads to the oxidation of tetrahydrobiopterin by nNOS-derived superoxide. Tetrahydrobiopterin is a known cofactor for NO synthesis by nNOS, binding near the heme and stabilizing the active dimeric structure of the enzyme. Tetrahydrobiopterin was found to reverse the guanabenz-mediated inhibition of nNOS in vitro. Similarly, administration of tetrahydrobiopterin to rats prevented both nNOS inhibition and loss of enzyme after guanabenz treatment, indicating that the loss of tetrahydrobiopterin plays a major role in the effects of guanabenz in vivo. To investigate if the loss of tetrahydrobiopterin was sufficient for eliciting the enhanced turnover of nNOS, we depleted tetrahydrobiopterin in cells by inhibiting GTP cyclohydrolase I with 2,4-diamino-6-hydroxypyrimidine. A 75% decrease in tetrahydrobiopterin levels led to a 2-fold increase in the amount of nNOS-ubiquitin conjugates detected. Consistent with our cellular observations, in vitro ubiquitination and degradation of nNOS by reticulocyte lysate proteins was decreased when tetrahydrobiopterin was added. Thus, tetrahydrobiopterin may serve as an endogenous regulator of nNOS protein levels. Through mutagenesis studies, we were able to localize the ubiquitination site to the calmodulin binding region of nNOS (residues 720-756). Peptide mapping studies using capillary flow liquid chromatography interfaced with a linear ion trap mass spectrometer identified residue 754 as a site for ubiquitin attachment. Furthermore, using methylated ubiquitin and purified nNOS, we determined that mono-ubiquitination of nNOS is sufficient for proteasomal degradation in vitro. Thus, it is possible that alterations of the heme active site structure, in this case through oxidation of tetrahydrobiopterin, are recognized by cellular factors that direct the ubiquitination of a lysine residue in the calmodulin binding region, resulting in the selective proteasomal degradation of nNOS.

Vesicles and micelle electrokinetic chromatography (EKC) were used to study solute partitioning from the aqueous phase into vesicles and micelles. The retention factors (k) of neutral solutes are related to their partition coefficients (K) and the phase ratio (f), k = K*f. Dihexadecyl phosphate (DHP) vesicles used in this study undergo gel to liquid-crystalline phase at the critical temperature. Investigation of thermodynamics of solute partitioning into bilayers would allow separating the enthalpic and entropic contributions to the free energy of transfer. Linear Solvation Energy Relationship (LSER) modeling was used to elucidate the contributions of hydrophobic, hydrogen bond interactions, dipolar, and polarizability to the free energy of transfer of solutes from the aqueous phase into the vesicle phase at above and below Tc. Hydrophobic interaction keeps an important role in solute partitioning into bilayers. Hydrogen bonding, electrostatic, and dipolar are also relative important. Polarity (p*) was directly measured at different locations in the DHP vesicles using different solvatochromic probes over a wide range of temperature. The size of liposomes and vesicles were also examined at different temperatures. In peptide mapping, models for the prediction of electrophoretic mobility and micelle-water partition coefficient of peptides were developed, and thus retention behavior of peptides, which should facilitate method development in MEKC. It allows rapid optimization of separation conditions that can lead to enhance resolution of complex mixtures. Initially, the partition coefficients of a training set of peptides were determined by MEKC. A quantitative structure-partition relationship (QSPR) was established based on the data set that relates partition coefficient (Kmc) to structural descriptors of the peptides. Also, a quantitative structure-migration relationship (QSMR) was developed based on the charge, residue mass, and length of peptide. An advantage of this model is that peptide descriptors can be calculated from the amino acid composition of the peptides. Different statistical methods were then used to determine the most relevant descriptors in the models and to test the accuracy and error for each model. The QSPR and QSMR models were then used to predict Kmc and peptide mobility for several membrane peptides and tryptic digest of horse cytochrome C.

The posttranslational modifications of core histones, such as the acetylation and methylation on lysine residues, play a critical role in a variety of gene activities such as transcription regulation, Deoxyribose Nucleic Acid (DNA) replication and damage repair, gene silencing and the regulation of cell developmental processes such as proliferation and differentiation. These modifications, as well as the responsible enzymes, can be related to the occurrence and development of many diseases such as leukemia, lupus and Huntington’s disease. A thorough investigation of these post-translational modification sites will shed light on the mechanism and function of histone modifications in these disorders.Traditional methods for the investigation of histone post-translational modifications are primarily immunoassay techniques. These techniques are very sensitive, but they rely greatly on the availability of the site specific antibodies. The lack of a comprehensive set of antibodies has limited the studies of the novel modification sites. An additional complication with immunoassays arises when multiple modifications occur on the same histone. The simultaneous presence of other modifications may impact the specificity of antibodies directed at a specific individual modification.In this study, mass spectrometry and its related techniques were applied for the investigation of histone post-translational modification. A model was first established to demonstrate that mass spectrometry and peptide mapping can be successfully applied to study the detailed localization of acetylation at specific lysine residues. Core histones were extracted from bovine thymus and identified by LC/MS. After the identification, these proteins were separated and digested using different enzymes. Direct peptide mapping was applied for these proteolytic digestion mixtures on a 7T FT ICR MS. The results showed more than 20 novel modification sites in addition to previously reported modification sites. Among them, the methylation of K59 in H4 was discovered to play a critical role in gene silencing.High Performance Liquid Chromatography Mass Spectrometry (LC/MS) peptide mapping was also performed for the confirmation of these novel modification sites on histone H4 peptide fragments digested by trypsin and pepsin. The results supported the observation of novel modification sites observed with Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (FT ICR MS). In addition, electron captured dissociation (ECD) was performed on H4 protein and its proteolytic digested fragments. Similar results were obtained as compared with peptide mapping.As an application, LC/MS was used to determine the differential expression of the histone modifications in Acute Myeloid Leukemia (AML) and Chronic Lymphocytic Leukemia (CLL). The inhibition in different cell lines and leukemia patients caused by two Histone Deacetylase (HDAC) inhibitors were determined. Simultaneous observation was made for multiple modifications in all core histone.Finally, another model was studied for quick identification and the semi-quantitative determination of histone acetylation. Deuterium-acetic anhydride (d6-acetic anhydride) was added to the partially acetylated protein/peptide for a D3-acetylation of the unmodified residues. D3-acetylation normalization was observed in peptide fragments containing lysine residues. The result is an easily identifiable isotope fingerprint that can be used as a “marker” for the investigation of the modification state of a particular fragment of the acetylated protein and peptide. Meanwhile, a simple comparison between the different D3-acetylated peak intensities will give us information regarding the composition of different modification isomers.