Suneel S. Apte

BACKGROUND: Dysregulated proteolysis is implicated in thoracic (thoracic aortic aneurysm [TAA]) and abdominal aortic aneurysm (AAA) pathogenesis, but proteolytic landscapes (degradomes) of aneurysmal and normal aorta and contributions of individual proteases remain undefined. Here, a proteome-wide approach was used to define and compare TAA and AAA degradomes and uncover the specific role in aortic remodeling of 2 proteases consistently identified in the aneurysms, CMA1 (mast cell chymase) and MMP9 (matrix metalloprotease 9). METHODS: The mass spectrometry-based N-terminomics strategy, terminal amine isotopic labeling of substrates, was applied to Marfan syndrome TAAs (n=5), AAAs (n=16), and nondiseased thoracic aorta (n=4), and abdominal aorta (n=4) in a forward degradomics application, that is, to define substrate and protease degradomes. 8-plex iTRAQ terminal amine isotopic labeling of substrates was used for quantitative comparison of the tissue cohorts. Cleavage sites of CMA1 and MMP9 were sought by reverse degradomics, that is, digestion of aortic proteins with these proteases, followed by terminal amine isotopic labeling of substrates. CMA1 and MMP9 proteolysis of biglycan was further resolved using amino-terminal oriented mass spectrometry of substrates. RESULTS: We experimentally annotated 20 885 proteolytically derived peptides and identified 129 proteases in the aortic tissues. Quantitative substrate degradome comparisons identified specific differentially modulated pathways and networks in TAAs and AAAs. Reverse degradomics elucidated >300 CMA1 and MMP9 substrate cleavage sites, of which many, including orthogonally validated biglycan cleavages, occurred in the disease degradomes. CONCLUSIONS: Unbiased forward degradomics of the aortic wall from TAA, AAA, and nondiseased tissue provides a systems biology view of aortic wall breakdown and a new resource for its hitherto occult proteolytic landscape, demonstrating widespread extracellular matrix remodeling with disproportionate impact on proteoglycans. The findings provided insights into aortic aneurysm pathways and disease biomarkers and suggest involvement of numerous proteases. Mapping of specific proteolytic contributions of CMA1 and MMP9 illustrates a strategy for defining the activities of all proteases involved in aortic disease.

The ADAMTS superfamily comprises 19 secreted metalloproteases (ADAMTS proteases) and seven structurally related secreted proteins without catalytic activity (ADAMTS-like proteins). Members of both families participate in diverse and numerous processes in a variety of tissues and organs during embryo development and in connective tissue maintenance and hemostasis in the adult. The functional relationships between the two families are unclear, although most ADAMTS roles can be attributed to proteolytic actions and most ADAMTS-like roles to their functions as matricellular proteins. ADAMTS proteases are implicated in human and animal Mendelian disorders and are key participants in acquired disease pathways, including inflammatory and degenerative conditions. Several spontaneous mutations and genetically engineered invertebrate and mammalian (mostly mouse) models have been used to uncover ADAMTS biological roles in organogenesis and to define their cellular impact and underlying mechanisms. Despite their profound biological and medical importance, detailed knowledge about ADAMTS molecular structures, substrates, or binding partners and biochemical mechanisms is still limited. ADAMTS2, ADAMTS5, and ADAMTS13 have previously been extensively investigated because of their obvious clinical relevance, but much less is known about other ADAMTS proteases and most ADAMTS-like proteins. Here, the essential background and concepts relevant to this superfamily of proteins are discussed.

Tissue breakdown, especially extracellular matrix or secretome breakdown is a significant aspect of physiological remodeling and disease processes in solid organs, with profound structural and regulatory impact. Tissue and circulating proteins undergo breakdown through a chemical process mediated by proteases, that is, hydrolysis of peptide bonds. Proteolysis has immense biological impact because it is irreversible, results in protein inactivation or activation, and can generate fragments with new functions to expand the functional genome. The traditional focus on a few proteases of interest against candidate substrates provided limited insights into the proteolytic landscape of human diseases. In contrast, innovations in protein terminomics workflows, tandem mass spectrometry and data handling now routinely permit identification of proteolytic events and proteases on the proteome scale, the degradome. The unbiased, de novo elucidation of disease degradomes, termed forward degradomics, has increased the number of known in vivo proteolytic events, despite only limited application to human disease to date. In reverse degradomics, activities of proteases are elucidated individually, but also at the proteome scale by digesting protein libraries sourced from tissues and cell secretomes, or by comparing the degradomes of protease‐deficient/overexpressing and parental cells. Integration of forward and reverse degradomes precisely defines protease primary mechanisms in disease. Cross‐disease degradome analysis can define disease‐relevant, protease‐specific biomarkers, identify proteases as appropriate therapeutic targets, and predict cross‐organ impact of protease inhibitors. A systematic effort to map disease degradomes, that is, a prospective human degradome project would generate a comprehensive proteolysis knowledgebase for diagnostics and therapeutics.

Proteolysis is an irreversible posttranslational modification with immense biological impact. Owing to its high disease significance, there is growing interest in investigating proteolysis on the proteome scale, termed degradomics. We developed ‘Database of Identified Cleavage sites Endemic to Disease states’ (DICED; https://diced.lerner.ccf.org/), as a searchable knowledgebase to promote collaboration and knowledge sharing in degradomics. DICED was designed and constructed using Python, JavaScript, HTML, and PostgreSQL. Django (https://www.djangoproject.com) was chosen as the primary framework for its security features and support for agile development. DICED can be utilized on major web browsers and operating systems for easy access to high‐throughput mass spectrometry‐identified cleaved protein termini. The data was obtained using N‐terminomics, comprising N‐terminal protein labeling, labeled peptide enrichment, mass spectrometry and positional peptide annotation. The DICED database contains experimentally derived N‐terminomics peptide datasets from tissues, diseases, or digests of tissue protein libraries using individual proteases and is searchable using UniProt ID, protein name, gene symbol or up to 100 peptide sequences. The tabular output format can be exported as a CSV file. Although DICED presently accesses data from a single laboratory, it is freely available as a Galaxy tool and the underlying database is scalable, permitting addition of new datasets and features.

The hypothalamic arcuate nucleus (ARH) contains neurons vital for maintaining energy homeostasis that sense and respond to changes in blood-borne metabolic hormones. Despite its juxtaposition to the median eminence (ME), a circumventricular organ lacking a blood-brain barrier and thus exposed to circulating molecules, only a few ventral ARH neurons perceive these extravasating metabolic signals due to a poorly understood ME/ARH diffusion barrier. Here, we show in male mice that aggrecan, a perineural-net proteoglycan deposited by orexigenic ARH neurons, creates a peculiar ventrodorsal diffusion gradient. Fasting enhances aggrecan deposition more dorsally, reinforcing the diffusion barrier, particularly around neurons adjacent to fenestrated capillary loops that enter the ARH. The disruption of aggrecan deposits results in unregulated diffusion of blood-borne molecules into the ARH and impairs food intake. Our findings reveal the molecular nature and plasticity of the ME/ARH diffusion barrier, and indicate its physiological role in hypothalamic metabolic hormone sensing.