Analysis of protein modifications: protein N-homocysteinylation and covalent inhibition of the LuxS enzyme by brominated furanones.
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http://hdl.handle.net/2047/d20002940
Beuning, Penny (Committee member)
Deth, Richard C. (Committee member)
Jones, Graham (Committee member)
Lee, David H. (Committee member)
Elevated blood levels of homocysteine (Hcy) have been established as an independent risk factor for cardiovascular disease and neurodegenerative disorders, such as Alzheimer's disease. Homocysteine-thiolactone (Hcy TL) is a metabolite of Hcy and reacts with the amino groups in proteins to form N-homocysteinylated (N-Hcy) proteins, which comprise a major pool of Hcy in humans. Protein N-homocysteinylation has been hypothesized as a contributing factor for the cytotoxic effects of elevated Hcy. Thus, there is an increasing interest in the systematic characterization of N-homocysteinylation and the discovery of related protein biomarkers. Due to the relatively low abundance of N-Hcy proteins, the detection of this posttranslational modification remains a major challenge in bioanalytical chemistry. In project 1, we report the development of several new chemical labeling methods to selectively derivatize N-Hcy proteins with various chemical tags, thereby facilitating subsequent proteomic analysis.
In order to demonstrate the advantages of chemical labeling methods, also called chemical derivatization, during analysis of protein posttranslational modifications, Chapter 1 describes the main detection techniques (e.g. antibody-based detection and mass spectrometry) for analysis of protein posttranslational modifications. It also details the application of chemical derivatization as an alternative method, which provides a basis for development of chemical methods for the detection of protein N-homocysteinylation.
Having shown the application of chemical derivatization for analysis of protein modifications, Chapter 2 details the development of different aldehyde tags (e.g. Rhodamine-aldehyde and biotin-aldehyde) to selectively label N-homocystamides to form 1,3-thiazine protein or peptide adducts which are suitable for mass spectrometric analysis. Under mild acidic conditions, γ-aminothiols in N-homocystamide groups irreversibly and stoichiometrically react with aldehydes to form stable 1,3-thiazines, whereas the reversible Schiff base formation between aldehydes and amino groups in native proteins is markedly disfavored due to protonation of amines. Higher detection sensitivity was reached by using biotin-aldehyde labeling followed by Western blotting and chemiluminescence detection. In addition, an enrichment method was also developed for capturing the low-abundance modified proteins using aldehyde resin to drastically reduce the sample complexity.
Following the method development, in Chapter 3, we further optimized the conditions for biotin-aldehyde labeling. After optimization, biotin-aldehyde labeling coupled with Western blotting and chemiluminescence detection was used for the quantification of changes in protein modifications in rat and mouse plasma and human serum under elevated homocysteine levels. In comparison to control samples, the overall levels of protein N-homocysteinylation were increased in rat plasma with vitamin B12 deficient diets and serum from male autistic children. More interestingly, the levels of modification varied significantly among individual protein bands. All together, this very first proteomic study reveals that protein modifications are affected by the blood levels of homocysteine, and furthermore, opens new avenues to better understand the underlying molecular mechanisms and to identify potential biomarkers for clinical diagnosis.
Chapters 2 and 3 are focused on the detection of protein N-homocysteinylation which is related to the metabolism of Hcy in mammals. Project 2 in Chapter 4 is related to a unique pathway of Hcy synthesis which is catalyzed by LuxS enzyme in bacteria. The LuxS enzyme plays very important roles in not only the metabolism of Hcy but also the induction of bacterial quorum sensing. In Chapter 4, the catalytic mechanism of LuxS and biomedical significance of quorum sensing and biofilm formation are first described. Quorum sensing has become a prospective drug target for the inhibition of bacterial biofilm formation. Antibacterial activities such as inhibition of biofilm formation and swarming have been reported for halogenated furanones, a class of natural products isolated from the marine red algae Delisea pulchra. These compounds exhibit low toxicity to mammalian cells; therefore, the furanones and their derivatives have served as lead compounds for the development of broad spectrum antibacterial agents. However, the molecular targets and the precise modes of action for furanones remain elusive. One attractive target is bacterial quorum sensing, the process by which bacteria sense and respond to population density, and consequently behave as a coordinated community. Quorum sensing involves the synthesis, secretion and detection of signaling molecules, commonly referred to as autoinducers (AIs). Previous work from our laboratory and others suggested that halogenated furanones disrupt the autoinducer 2 (AI-2) biosynthetic pathway. Based on that, in Chapter 4, we investigated the inhibition activities and detailed inhibition mechanism of brominated furanones against LuxS enzyme which catalyzes production of AI-2. We find biochemical and chemical evidence that brominated furanones inactivate the AI-2 producing enzyme LuxS by covalently modifying cysteine 126 in LuxS following a direct addition/elimination mechanism. The results also show that the LuxS enzyme is a potential target of brominated furanones for the inhibition of bacterial quorum sensing and the development of furanone derivatives will provide a new direction for the inhibition of biofilm and bacterial infection.
Based on the described work, Hcy metabolism is very important for both eukaryotes and prokaryotes. In Chapter 5, the future directions for both projects are evaluated. For the detection of protein N-homocysteinylation, the chemical methods will be further optimized and the work will still focus on the identification of N-Hcy proteins which may become potential biomarkers associated with homocysteinemia for disease diagnosis. For the inhibition of brominated furanones against quorum sensing, design of furanone derivatives with low cytotoxicity will be recommended for development of new furanone-based antibiotics against biofilm formation.
Chemical derivatization
LuxS enzyme
Protein N-homocysteinylation
Protein posttranslational modification
quorum sensing
Analytical Chemistry
Chemistry
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