Analytical Strategies for the Identification and Characterization of RAGE Binders of Proinflammatory mediators. AGEs and ALES
Tesi di Dottorato
Data di Pubblicazione:
2019
Citazione:
Analytical Strategies for the Identification and Characterization of RAGE Binders of Proinflammatory mediators. AGEs and ALES / M.h.a. Mol ; tutor: G. Aldini ; phd coordinator: G. Aldini. DIPARTIMENTO DI SCIENZE FARMACEUTICHE, 2019 Sep 16. 31. ciclo, Anno Accademico 2018. [10.13130/mol-marco-hendrikus-adrianus_phd2019-09-16].
Abstract:
INTRODUCTION
AGEs and ALEs (Advanced Glycoxidation/Lipoxidation End products) are covalently modified proteins that can act as pathogenic factors in several chronic diseases, like diabetes and cardiovascular diseases. These covalent adducts are formed by different mechanisms. AGEs are proteins covalently modified by reducing sugars or their oxidative degradation products, involving the Maillard reaction. ALEs are proteins modified by reactive carbonyl species (RCS) generated by lipid peroxidation. AGEs/ALEs can be the basis of many different pathologies, underlining the importance for good analytical methods for identification and characterization for the use of biomarkers, but also as a drug target. However, the identification, characterization and quantification of AGEs/ALEs remains to be very challenging due to heterogeneous precursors (sugars, lipids) leading to heterogeneous AGEs/ALEs, present in low concentrations and being very complex analytes. Various techniques to identify and characterize AGEs/ALEs have been described, making use of an isolation/enrichment step based on reactive groups, like carbonyls. However, not all AGEs/ALEs retain reactive groups and therefore can not be isolated and identified using these techniques, indicating the need for a new strategy. The strategy that has been employed in our laboratory is to use the soluble domain of the RAGE receptor, VC1, to affinity enrich AGEs. Using this approach, AGEs/ALEs will be enriched independently of the protein and type of modification. Moreover, a ligand of RAGE can be identified, which could be a potential biomarker of a disease caused by oxidative stress.
RAGE is a type I cell surface receptor that is expressed in several cells, such as endothelial cells, smooth muscle cells, but also dendritic cells and T-lymphocytes and is predominantly located in the lungs. The receptor has been implicated in many different pathologies with a marked oxidative base, such as diabetes, atherosclerosis and neurodegenerative diseases. One of the pathways that can be activated is the Nf-κB pathway. The Nf-κB pathway is the ideal signaling pathway to investigate the binding and activation of RAGE by AGEs or ALEs. For this purpose, a cell line was obtained with and without overexpression of RAGE. Furthermore, the cell lines were transfected with a Nf-κB reporter gene, providing us with a fast and high-throughput assay for the evaluation of a pro-inflammatory response upon stimulation with AGEs/ALEs.
AIM OF THE PROJECT
The identification and characterization of AGEs/ALEs has proven to be crucial in the onset and development of many pathologies. Therefore, good analytical strategies need to be developed/optimized for better understanding of the exact nature of modification, to understand the role they play in disease progression. Identified AGEs/ALEs can serve as biomarker, as well as drug targets. The VC1 technique was proven to be a promising technique to accommodate the need for enrichment of AGEs for better characterization. The first aim of the project was therefore to investigate whether also ALEs are binder of RAGE, since they share the same structural properties than AGEs, and also have been shown to activate the Nf-κB pathway, implicating a role for receptors, like RAGE. Furthermore, to gain a deeper insight into the molecular mechanisms involved in the protein-protein engagement.
Since a successful enrichment strategy was developed, the second aim of this project was focused on identifying AGEs/ALEs in biological samples. The first part was focused on oxidizing healthy human plasma in-vitro using AAPH as a radical initiator, and the incubation of plasma directly with RCS, anticipating the production of AGEs/ALEs. The VC1 technique was then used to identify whic
Tipologia IRIS:
Tesi di dottorato
Keywords:
Oxidation; advanced glycation end products; advanced lipoxidation end products; affinity chromatography; mass spectrometry; receptor for advanced glycation endproducts; NF-kB activity
Elenco autori:
M.H.A. Mol
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