RESUMEN
The N-terminomics approach of Terminal Amine Isotopic Labeling of Substrates (TAILS) enables the identification and quantification of natural and neo-N-termini of proteins using liquid chromatography and tandem mass spectrometry (LC-MS/MS). This methodology has been used to study protease function and identify protease substrates in cell culture systems, animal disease models, and more recently, has been applied to clinical samples. Here, we present the application of TAILS to tissue and liquid biopsies.
Asunto(s)
Aminas , Proteómica , Aminas/química , Animales , Cromatografía Liquida , Marcaje Isotópico/métodos , Biopsia Líquida , Péptido Hidrolasas/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Espectrometría de Masas en Tándem/métodosRESUMEN
Dysregulated protease activity is often implicated in the initiation of inflammation and immune cell recruitment in gastrointestinal inflammatory diseases. Using N-terminomics/TAILS (terminal amine isotopic labeling of substrates), we compared proteases, along with their substrates and inhibitors, between colonic mucosal biopsies of healthy patients and those with ulcerative colitis (UC). Among the 1642 N-termini enriched using TAILS, increased endogenous processing of proteins was identified in UC compared to healthy patients. Changes in the reactome pathways for proteins associated with metabolism, adherens junction proteins (E-cadherin, liver-intestinal cadherin, catenin alpha-1, and catenin delta-1), and neutrophil degranulation were identified between the two groups. Increased neutrophil infiltration and distinct proteases observed in ulcerative colitis may result in extensive break down, altered processing, or increased remodeling of adherens junctions and other cellular functions. Analysis of the preferred proteolytic cleavage sites indicated that the majority of proteolytic activity and processing comes from host proteases, but that key microbial proteases may also play a role in maintaining homeostasis. Thus, the identification of distinct proteases and processing of their substrates improves the understanding of dysregulated proteolysis in normal intestinal physiology and ulcerative colitis.
Asunto(s)
Colitis Ulcerosa/fisiopatología , Péptido Hidrolasas/metabolismo , Inhibidores de Proteasas/metabolismo , Proteolisis , Proteómica/métodos , Adulto , Anciano , Secuencia de Aminoácidos , Sitios de Unión , Biopsia , Cadherinas/metabolismo , Cateninas/metabolismo , Cromatografía Líquida de Alta Presión , Colon/patología , Femenino , Humanos , Marcaje Isotópico/métodos , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Péptidos/análisis , Unión Proteica , Transducción de SeñalRESUMEN
Matrix metalloproteinases (MMPs) have been studied in the context of cancer due to their ability to increase cell invasion, and were initially thought to facilitate metastasis solely through the degradation of the extracellular matrix (ECM). MMPs have also been investigated in the context of their ECM remodeling activity in several acute and chronic inflammatory diseases. However, after several MMP inhibitors failed in phase III clinical trials, a global reassessment of their biological functions was undertaken, which has revealed multiple unanticipated functions including the processing of chemokines, cytokines, and cell surface receptors. Despite what their name suggests, the matrix aspect of MMPs could contribute to a lesser part of their physiological functions in inflammatory diseases, as originally anticipated. Here, we present examples of MMP substrates implicated in cell signaling, independent of their ECM functions, and discuss the impact for the use of MMP inhibitors.