Abstract
Both protease- and reactive oxygen species (ROS)-mediated proteolysis are thought to be key effectors of tissue remodeling. We have previously shown that comparison of amino acid composition can predict the differential susceptibilities of proteins to photo-oxidation. However, predicting protein susceptibility to endogenous proteases remains challenging. Here, we aim to develop bioinformatics tools to (i) predict cleavage site locations (and hence putative protein susceptibilities) and (ii) compare the predicted vulnerabilities of skin proteins to protease- and ROS-mediated proteolysis. The first goal of this study was to experimentally evaluate the ability of existing protease cleavage site prediction models (PROSPER and DeepCleave) to identify experimentally determined MMP9 cleavage sites in two purified proteins and in a complex human dermal fibroblast-derived extracellular matrix (ECM) proteome. We subsequently developed deep bidirectional recurrent neural network (BRNN) models to predict cleavage sites for 14 tissue proteases. The predictions of the new models were tested against experimental datasets and combined with amino acid composition analysis (to predict ultraviolet radiation (UVR)/ROS susceptibility) in a new web app: the Manchester proteome susceptibility calculator (MPSC). The BRNN models performed better in predicting cleavage sites in native dermal ECM proteins than existing models (DeepCleave and PROSPER), and application of MPSC to the skin proteome suggests that: compared with the elastic fiber network, fibrillar collagens may be susceptible primarily to protease-mediated proteolysis. We also identify additional putative targets of oxidative damage (dermatopontin, fibulins and defensins) and protease action (laminins and nidogen). MPSC has the potential to identify potential targets of proteolysis in disparate tissues and disease states.
Highlights
The causative mechanisms of aging are not yet fully understood, there is compelling evidence that biochemical pathways, including protein oxidation, and proteasemediated cleavage, contribute to proteolysis and loss of proteostasis [1]
For example, ultraviolet radiation (UVR), oxidative stress, and upregulated protease activity are interlinked processes associated with clinical photoaging, which manifests in the dermis as profound histological remodeling of fibrillar collagens and elastic fibers and the accumulation of oxidative damage [5,6,7,8]
As the dataset is highly imbalanced, containing thousands of non-cleavage sites vs. only hundreds of cleavage sites, and in order to evaluate the performance of PROSPER, DeepCleave and MPC, we used F1 score representing the harmonic mean of precision and recall; the Matthews’ correlation coefficient (MCC), precision, recall and area under the curve (AUC) scores
Summary
The causative mechanisms of aging are not yet fully understood, there is compelling evidence that biochemical pathways, including protein oxidation, and proteasemediated cleavage, contribute to proteolysis and loss of proteostasis [1]. Previous studies from our group suggest that both the in vivo remodeling of elastic fiber-associated fibrillin microfibrils, which is a hallmark of early photoaging, and the relative susceptibility of these assemblies to in vitro UVR, is likely to be due to specific amino acid (AA) compositions of the component proteins, principally fibrillin-1 [7,12,13]. This protein, and others enriched in both UVR-absorbing (UV-chromophore) and oxidationsensitive amino acid (AA) residues, are susceptible to degradation by environmentally attainable UVR doses [14]. There remains the possibility that (as with relative UVR chromophore abundance) protease cleavage site abundance may serve as a key determinant of relative protease susceptibility
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