RESUMEN
Extracellular proteolysis critically regulates cellular and tissue responses and is often dysregulated in human diseases. The crosstalk between proteolytic processing and other major post-translational modifications (PTMs) is emerging as an important regulatory mechanism to modulate protease activity and maintain cellular and tissue homeostasis. Here, we focus on matrix metalloproteinase (MMP)-mediated cleavages and N-acetylgalactosamine (GalNAc)-type of O-glycosylation, two major PTMs of proteins in the extracellular space. We investigated the influence of truncated O-glycan trees, also referred to as Tn antigen, following the inactivation of C1GALT1-specific chaperone 1 (COSMC) on the general and MMP9-specific proteolytic processing in MDA-MB-231 breast cancer cells. Quantitative assessment of the proteome and N-terminome using terminal amine isotopic labelling of substrates (TAILS) technology revealed enhanced proteolysis by MMP9 within the extracellular proteomes of MDA-MB-231 cells expressing Tn antigen. In addition, we detected substantial modifications in the proteome and discovered novel ectodomain shedding events regulated by the truncation of O-glycans. These results highlight the critical role of mature O-glycosylation in fine-tuning proteolytic processing and proteome homeostasis by modulating protein susceptibility to proteolytic degradation. These data suggest a complex interplay between proteolysis and O-GalNAc glycosylation, possibly affecting cancer phenotypes.
Asunto(s)
Proteolisis , Humanos , Glicosilación , Línea Celular Tumoral , Metaloproteinasa 9 de la Matriz/metabolismo , Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Galactosiltransferasas/metabolismo , Galactosiltransferasas/genética , Procesamiento Proteico-Postraduccional , Proteoma/metabolismo , Proteoma/análisis , Chaperonas MolecularesRESUMEN
Positional proteomics methodologies have transformed protease research, and have brought mass spectrometry (MS)-based degradomics studies to the forefront of protease characterization and system-wide interrogation of protease signaling. Considerable advancements in both sensitivity and throughput of liquid chromatography (LC)-MS/MS instrumentation enable the generation of enormous positional proteomics datasets of natural and protein termini and neo-termini of cleaved protease substrates. However, concomitant progress has not been observed to the same extent in data analysis and post-processing steps, arguably constituting the largest bottleneck in positional proteomics workflows. Here, we present a computational tool, CLIPPER 2.0, that builds on prior algorithms developed for MS-based protein termini analysis, facilitating peptide-level annotation and data analysis. CLIPPER 2.0 can be used with several sample preparation workflows and proteomics search algorithms and enables fast and automated database information retrieval, statistical and network analysis, as well as visualization of terminomic datasets. We demonstrate the applicability of our tool by analyzing GluC and MMP9 cleavages in HeLa lysates. CLIPPER 2.0 is available at https://github.com/UadKLab/CLIPPER-2.0.
Asunto(s)
Péptidos , Proteómica , Espectrometría de Masas en Tándem , Proteómica/métodos , Humanos , Péptidos/metabolismo , Péptidos/análisis , Células HeLa , Espectrometría de Masas en Tándem/métodos , Algoritmos , Programas Informáticos , Bases de Datos de Proteínas , Cromatografía Liquida , Anotación de Secuencia Molecular , Análisis de Datos , Metaloproteinasa 9 de la Matriz/metabolismoRESUMEN
Proteases have long been associated with cancer progression, due to their ability to facilitate invasion upon matrix remodelling. However, proteases are not simply degraders of the matrix, but also play fundamental roles in modulating cellular behaviour through the proteolytic processing of specific substrates. Indeed, proteases can elicit both pro- and anti- tumorigenic effects depending on context. Using a heterocellular spheroid model of breast cancer progression, we demonstrate the repressive function of myoepithelial ADAMTS3, with its loss directing myoepithelial-led invasion of luminal cells through a physiologically relevant matrix. Degradomic analysis, using terminal amine isotopic labelling of substrates (TAILS), combined with functional assays, implicate ADAMTS3 as a mediator of fibronectin degradation. We show further that loss of ADAMTS3 enhances levels of fibronectin in the microenvironment, promoting invasion through canonical integrin α5ß1 activation. Our data highlight a tumour suppressive role for ADAMTS3 in early stage breast cancer, and contribute to the growing evidence that proteases can restrain cancer progression.
Asunto(s)
Neoplasias de la Mama , Femenino , Humanos , Mama , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Fibronectinas/genética , Fibronectinas/metabolismo , Péptido Hidrolasas/metabolismo , Microambiente TumoralRESUMEN
Vertebrate lonesome kinase (VLK) is the only known secreted tyrosine kinase and responsible for the phosphorylation of a broad range of secretory pathway-resident and extracellular matrix proteins. However, its cell-type specific functions in vivo are still largely unknown. Therefore, we generated mice lacking the VLK gene (protein kinase domain containing, cytoplasmic (Pkdcc)) in mesenchymal cells. Most of the homozygous mice died shortly after birth, most likely as a consequence of their lung abnormalities and consequent respiratory failure. E18.5 embryonic lungs showed a reduction of alveolar type II cells, smaller bronchi, and an increased lung tissue density. Global mass spectrometry-based quantitative proteomics identified 97 proteins with significantly and at least 1.5-fold differential abundance between genotypes. Twenty-five of these had been assigned to the extracellular region and 15 to the mouse matrisome. Specifically, fibromodulin and matrilin-4, which are involved in extracellular matrix organization, were significantly more abundant in lungs from Pkdcc knockout embryos. These results support a role for mesenchyme-derived VLK in lung development through regulation of matrix dynamics and the resulting modulation of alveolar epithelial cell differentiation.
Asunto(s)
Matriz Extracelular , Proteínas Quinasas , Animales , Ratones , Proteínas Quinasas/genética , Organogénesis/genética , Pulmón , Mesodermo , Vertebrados , Proteínas Tirosina QuinasasRESUMEN
Proteases modulate critical processes in cutaneous tissue repair to orchestrate inflammation, cell proliferation and tissue remodeling. However, the functional consequences and implications in healing impairments of most cleavage events are not understood. Using iTRAQ-based Terminal Amine Isotopic Labeling of Substrates (TAILS) we had characterized proteolytic signatures in a porcine wound healing model and identified two neo-N termini derived from proteolytic cleavage of the focal adhesion protein and mechanotransducer zyxin. Here, we assign these proteolytic events to the activity of either caspase-1 or serine protease HtrA1 and analyze the biological relevance of the resultant zyxin truncations. By cellular expression of full-length and truncated zyxin proteins, we demonstrate nuclear translocation of a C-terminal zyxin fragment that could also be generated in vitro by HtrA1 cleavage and provide evidence for its anti-apoptotic activities, potentially by regulating the expression of modulators of cell proliferation, protein synthesis and genome stability. Targeted degradomics correlated endogenous generation of the same zyxin fragment with increased cell density in human primary dermal fibroblasts. Hence, this newly identified HtrA1-zyxin protease signaling axis might present a novel mechanism to transiently enhance cell survival in environments of increased cell density like in wound granulation tissue.
Asunto(s)
Serina Peptidasa A1 que Requiere Temperaturas Altas/metabolismo , Zixina/metabolismo , Recuento de Células , Núcleo Celular/metabolismo , Supervivencia Celular , Células HeLa , Serina Peptidasa A1 que Requiere Temperaturas Altas/fisiología , Humanos , Péptido Hidrolasas/metabolismo , Procesamiento Proteico-Postraduccional , Proteolisis , Proteoma/metabolismo , Proteómica , Piel/metabolismo , Células THP-1 , Zixina/fisiologíaRESUMEN
Due to their capacity to process different proteins of the extracellular matrix (ECM), matrix metalloproteinases (MMPs) were initially described as a family of secreted proteases, functioning as main ECM regulators. However, through proteolytic processing of various biomolecules, MMPs also modulate intra- and extracellular pathways and networks. Thereby, they are functionally implicated in the regulation of multiple physiological and pathological processes. Consequently, MMP activity is tightly regulated through a combination of epigenetic, transcriptional, and post-transcriptional control of gene expression, proteolytic activation, post-translational modifications (PTMs), and extracellular inhibition. In addition, MMPs, their substrates and ECM binding partners are frequently modified by PTMs, which suggests an important role of PTMs in modulating the pleiotropic activities of these proteases. This review summarizes the recent progress towards understanding the role of PTMs (glycosylation, phosphorylation, glycosaminoglycans) on the activity of several members of the MMP family.
Asunto(s)
Metaloproteinasas de la Matriz/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Activación Enzimática , Matriz Extracelular/metabolismo , Regulación de la Expresión Génica , Humanos , Metaloproteinasas de la Matriz/genética , Proteolisis , Especificidad por SustratoRESUMEN
Global characterization of protein N termini provides valuable information on proteome dynamics and diversity in health and disease. Driven by the progress in mass spectrometry-based proteomics, novel approaches for the dedicated investigation of protein N termini and protease substrates have been recently developed. Terminal amine isotopic labeling of substrates (TAILS) is a quantitative proteomics approach suitable for high-throughput and system-wide profiling of protein N termini in complex biological matrices. TAILS employs isotopic labeling of primary amines of intact proteins in combination with an amine-reactive high molecular weight polymer (HPG-ALD) for depletion of internal tryptic peptides and high enrichment of protein N termini by negative selection. Thereby, TAILS allows simultaneous identification of the natural N termini, protease-generated neo-N termini, and endogenously modified (e.g., acetylated) N termini. In this chapter, we provide a protocol for tandem mass tag (TMT)-TAILS analysis and further discuss specific considerations regarding N-terminome data interpretation using Proteome Discoverer™ software.