Substrate O-glycosylation actively regulates extracellular proteolysis.

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.

CLIPPER 2.0: Peptide-Level Annotation and Data Analysis for Positional Proteomics.

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.

Redefining metalloproteases specificity through network proteolysis.

Proteolytic processes on cell surfaces and extracellular matrix (ECM) sustain cell behavior and tissue integrity in health and disease. Matrix metalloproteases (MMPs) and a disintegrin and metalloproteases (ADAMs) remodel cell microenvironments through irreversible proteolysis of ECM proteins and cell surface bioactive molecules. Pan-MMP inhibitors in inflammation and cancer clinical trials have encountered challenges due to promiscuous activities of MMPs. Systems biology advances revealed that MMPs initiate multifactorial proteolytic cascades, creating new substrates, activating or suppressing other MMPs, and generating signaling molecules. This review highlights the intricate network that underscores the role of MMPs beyond individual substrate-enzyme activities. Gaining insight into MMP function and tissue specificity is crucial for developing effective drug discovery strategies and novel therapeutics. This requires considering the dynamic cellular processes and consequences of network proteolysis.

TGFß-mediated MMP13 secretion drives myoepithelial cell dependent breast cancer progression.

Ductal carcinoma in situ (DCIS) is a non-obligate precursor of invasive breast cancer. Virtually all women with DCIS are treated, despite evidence suggesting up to half would remain with stable, non-threatening, disease. Overtreatment thus presents a pressing issue in DCIS management. To understand the role of the normally tumour suppressive myoepithelial cell in disease progression we present a 3D in vitro model incorporating both luminal and myoepithelial cells in physiomimetic conditions. We demonstrate that DCIS-associated myoepithelial cells promote striking myoepithelial-led invasion of luminal cells, mediated by the collagenase MMP13 through a non-canonical TGFß - EP300 pathway. In vivo, MMP13 expression is associated with stromal invasion in a murine model of DCIS progression and is elevated in myoepithelial cells of clinical high-grade DCIS cases. Our data identify a key role for myoepithelial-derived MMP13 in facilitating DCIS progression and point the way towards a robust marker for risk stratification in DCIS patients.

Longitudinal Evaluation of Biomarkers in Wound Fluids from Venous Leg Ulcers and Split-thickness Skin Graft Donor Site Wounds Treated with a Protease-modulating Wound Dressing.

Venous leg ulcers represent a clinical challenge and impair the quality of life of patients. This study examines impaired wound healing in venous leg ulcers at the molecular level. Protein expression patterns for biomarkers were analysed in venous leg ulcer wound fluids from 57 patients treated with a protease-modulating polyacrylate wound dressing for 12 weeks, and compared with exudates from 10 acute split-thickness wounds. Wound healing improved in the venous leg ulcer wounds: 61.4% of the 57 patients with venous leg ulcer achieved a relative wound area reduction of ≥ 40%, and 50.9% of the total 57 patients achieved a relative wound area reduction of ≥ 60%. Within the first 14 days, abundances of S100A8, S100A9, neutrophil elastase, matrix metalloproteinase-2, and fibronectin in venous leg ulcer exudates decreased significantly and remained stable, yet higher than in acute wounds. Interleukin-1ß, tumour necrosis factor alpha, and matrix metalloproteinase-9 abundance ranges were similar in venous leg ulcers and acute wound fluids. Collagen (I) α1 abundance was higher in venous leg ulcer wound fluids and was not significantly regulated. Overall, significant biomarker changes occurred in the first 14 days before a clinically robust healing response in the venous leg ulcer cohort.

Degradomics technologies in matrisome exploration.

Consisting of a defined set of extracellular proteins secreted from resident cells and with minor contributions from serum proteins, the extracellular matrix (ECM) is an essential component of all tissues. Maintaining tissue homeostasis, structural support and cellular control through cell-ECM communication, the ECM has come to be viewed as not just a passive structural entity but rather as a dynamic signaling conduit between cells and the extracellular compartment. Proteins and their cleavage products mediate this communication, and aberrant signaling, either directly or indirectly distorting the ECM, results in pathological conditions including cancer, inflammation, fibrosis, and neurodegenerative diseases. Characterization of ECM components, the matrisome, the extracellular environment and their changes in disease is therefore of importance to understand and mitigate by developing novel therapeutics. Liquid chromatography-mass spectrometry (LC-MS) proteomics has been integral to protein and proteome research for decades and long superseded the obsolescent gel-based approaches. A continuous effort has ensured progress with increased sensitivity and throughput as more advanced equipment has been developed hand in hand with specialized enrichment, detection, and identification methods. Part of this effort lies in the field of degradomics, a branch of proteomics focused on discovering novel protease substrates by identification of protease-generated neo-N termini, the N-terminome, and characterizing the responsible protease networks. Various methods to do so have been developed, some specialized for specific tissue types, others for particular proteases, throughput, or ease of use. This review aims to provide an overview of the state-of-the-art proteomics techniques that have successfully been recently utilized to characterize proteolytic cleavages in the ECM and thereby guided new research and understanding of the ECM and matrisome biology.

ADAM17-mediated EGFR ligand shedding directs macrophage-promoted cancer cell invasion.

Macrophages in the tumor microenvironment have a substantial impact on tumor progression. Depending on the signaling environment in the tumor, macrophages can either support or constrain tumor progression. It is therefore of therapeutic interest to identify the tumor-derived factors that control macrophage education. With this aim, we correlated the expression of A Disintegrin and Metalloproteinase (ADAM) proteases, which are key mediators of cell-cell signaling, to the expression of protumorigenic macrophage markers in human cancer cohorts. We identified ADAM17, a sheddase upregulated in many cancer types, as a protein of interest. Depletion of ADAM17 in cancer cell lines reduced the expression of several protumorigenic markers in neighboring macrophages in vitro as well as in mouse models. Moreover, ADAM17-/- educated macrophages demonstrated a reduced ability to induce cancer cell invasion. Using mass spectrometry-based proteomics and ELISA, we identified heparin-binding EGF (HB-EGF) and amphiregulin, shed by ADAM17 in the cancer cells, as the implicated molecular mediators of macrophage education. Additionally, RNA-Seq and ELISA experiments revealed that ADAM17-dependent HB-EGF ligand release induced the expression and secretion of CXCL chemokines in macrophages, which in turn stimulated cancer cell invasion. In conclusion, we provide evidence that ADAM17 mediates a paracrine EGFR-ligand-chemokine feedback loop, whereby cancer cells hijack macrophages to promote tumor progression.

Mapping specificity, cleavage entropy, allosteric changes and substrates of blood proteases in a high-throughput screen.

Proteases are among the largest protein families and critical regulators of biochemical processes like apoptosis and blood coagulation. Knowledge of proteases has been expanded by the development of proteomic approaches, however, technology for multiplexed screening of proteases within native environments is currently lacking behind. Here we introduce a simple method to profile protease activity based on isolation of protease products from native lysates using a 96FASP filter, their analysis in a mass spectrometer and a custom data analysis pipeline. The method is significantly faster, cheaper, technically less demanding, easy to multiplex and produces accurate protease fingerprints. Using the blood cascade proteases as a case study, we obtain protease substrate profiles that can be used to map specificity, cleavage entropy and allosteric effects and to design protease probes. The data further show that protease substrate predictions enable the selection of potential physiological substrates for targeted validation in biochemical assays.

Unraveling the structure and function of CdcPDE: A novel phosphodiesterase from Crotalus durissus collilineatus snake venom.

This study reports the isolation, structural, biochemical, and functional characterization of a novel phosphodiesterase from Crotalus durissus collilineatus venom (CdcPDE). CdcPDE was successfully isolated from whole venom using three chromatographic steps and represented 0.7% of total protein content. CdcPDE was inhibited by EDTA and reducing agents, demonstrating that metal ions and disulfide bonds are necessary for its enzymatic activity. The highest enzymatic activity was observed at pH 8-8.5 and 37 °C. Kinetic parameters indicated a higher affinity for the substrate bis(p-nitrophenyl) phosphate compared to others snake venom PDEs. Its structural characterization was done by the determination of the protein primary sequence by Edman degradation and mass spectrometry, and completed by the building of molecular and docking-based models. Functional in vitro assays showed that CdcPDE is capable of inhibiting platelet aggregation induced by adenosine diphosphate in a dose-dependent manner and demonstrated that CdcPDE is cytotoxic to human keratinocytes. CdcPDE was recognized by the crotalid antivenom produced by the Instituto Butantan. These findings demonstrate that the study of snake venom toxins can reveal new molecules that may be relevant in cases of snakebite envenoming, and that can be used as molecular tools to study pathophysiological processes due to their specific biological activities.

Matrix Metalloproteinases: How Much Can They Do?

Zinc-dependent matrix metalloproteinases (MMPs) belong to metzincins that comprise not only 23 human MMPs but also other metalloproteinases, such as 21 human ADAMs (a disintegrin and metalloproteinase domain) and 19 secreted ADAMTSs (a disintegrin and metalloproteinase thrombospondin domain). The many setbacks from the clinical trials of broad-spectrum MMP inhibitors for cancer indications in the late 1990s emphasized the extreme complexity of the participation of these proteolytic enzymes in biology. This editorial mini-review summarizes the Special Issue, which includes four review articles and 10 original articles that highlight the versatile roles of MMPs, ADAMs, and ADAMTSs, in normal physiology as well as in neoplastic and destructive processes in tissue. In addition, we briefly discuss the unambiguous involvement of MMPs in wound healing.

Mapping the N-Terminome in Tissue Biopsies by PCT-TAILS.

Proteases play pivotal roles in multiple biological processes in all living organisms and are tightly regulated under normal conditions, but alterations in the proteolytic system and uncontrolled protease activity result in multiple pathological conditions. A disease will most often be defined by an ensemble of cleavage events-a proteolytic signature, thus the system-wide study of protease substrates has gained significant attention and identification of disease specific clusters of protease substrates holds great promise as targets for diagnostics and therapy.In this chapter we describe a method that enables fast and reproducible analysis of protease substrates and proteolytic products in an amount of tissue less than the quantity obtained by a standard biopsy. The method combines tissue disruption and protein extraction by pressure cycling technology (PCT), N-terminal enrichment by tandem mass tag (TMT)-terminal amine isotopic labeling of substrates (TAILS), peptide analysis by mass spectrometry (MS), and a general pipeline for interpretation of the data.

Protease Activity Profiling of Snake Venoms Using High-Throughput Peptide Screening.

Snake venom metalloproteinases (SVMPs) and snake venom serine proteinases (SVSPs) are among the most abundant enzymes in many snake venoms, particularly among viperids. These proteinases are responsible for some of the clinical manifestations classically seen in viperid envenomings, including hemorrhage, necrosis, and coagulopathies. The objective of this study was to investigate the enzymatic activities of these proteins using a high-throughput peptide library to screen for the proteinase targets of the venoms of five viperid (Echis carinatus, Bothrops asper, Daboia russelii, Bitis arietans, Bitis gabonica) and one elapid (Naja nigricollis) species of high medical importance. The proteinase activities of these venoms were each tested against 360 peptide substrates, yielding 2160 activity profiles. A nonlinear regression model that accurately described the observed enzymatic activities was fitted to the experimental data, allowing for the comparison of cleavage rates across species. In this study, previously unknown protein targets of snake venom proteinases were identified, potentially implicating novel human and animal proteins that may be involved in the pathophysiology of viper envenomings. The functional relevance of these targets was further evaluated and discussed. These new findings may contribute to our understanding of the clinical manifestations and underlying biochemical mechanisms of snakebite envenoming by viperid species.

Large-Scale Quantitative Proteomics Identifies the Ubiquitin Ligase Nedd4-1 as an Essential Regulator of Liver Regeneration.

The liver is the only organ in mammals that fully regenerates even after major injury. To identify orchestrators of this regenerative response, we performed quantitative large-scale proteomics analysis of cytoplasmic and nuclear fractions from normal versus regenerating mouse liver. Proteins of the ubiquitin-proteasome pathway were rapidly upregulated after two-third hepatectomy, with the ubiquitin ligase Nedd4-1 being a top hit. In vivo knockdown of Nedd4-1 in hepatocytes through nanoparticle-mediated delivery of small interfering RNA caused severe liver damage and inhibition of cell proliferation after hepatectomy, resulting in liver failure. Mechanistically, we demonstrate that Nedd4-1 is required for efficient internalization of major growth factor receptors involved in liver regeneration and their downstream mitogenic signaling. These results highlight the power of large-scale proteomics to identify key players in liver regeneration and the importance of posttranslational regulation of growth factor signaling in this process. Finally, they identify an essential function of Nedd4-1 in tissue repair.

TAILS N-Terminomics and Proteomics Show Protein Degradation Dominates over Proteolytic Processing by Cathepsins in Pancreatic Tumors.

Deregulated cathepsin proteolysis occurs across numerous cancers, but in vivo substrates mediating tumorigenesis remain ill-defined. Applying 8-plex iTRAQ terminal amine isotopic labeling of substrates (TAILS), a systems-level N-terminome degradomics approach, we identified cathepsin B, H, L, S, and Z in vivo substrates and cleavage sites with the use of six different cathepsin knockout genotypes in the Rip1-Tag2 mouse model of pancreatic neuroendocrine tumorigenesis. Among 1,935 proteins and 1,114 N termini identified by TAILS, stable proteolytic products were identified in wild-type tumors compared with one or more different cathepsin knockouts (17%-44% of 139 cleavages). This suggests a lack of compensation at the substrate level by other cathepsins. The majority of neo-N termini (56%-83%) for all cathepsins was consistent with protein degradation. We validated substrates, including the glycolytic enzyme pyruvate kinase M2 associated with the Warburg effect, the ER chaperone GRP78, and the oncoprotein prothymosin-alpha. Thus, the identification of cathepsin substrates in tumorigenesis improves the understanding of cathepsin functions in normal physiology and cancer.

Autocrine and Paracrine Regulation of Keratinocyte Proliferation through a Novel Nrf2-IL-36γ Pathway.

The Nrf2 transcription factor is well known for its cytoprotective functions through regulation of genes involved in the detoxification of reactive oxygen species or toxic compounds. Therefore, activation of Nrf2 is a promising strategy for the protection of tissues from various types of insults and for cancer prevention. However, recent studies revealed a proinflammatory activity of activated Nrf2 and a stimulating effect on epithelial cell proliferation, but the underlying mechanisms of action and the responsible target genes are largely unknown. Using a combination of gene expression profiling, chromatin immunoprecipitation, and targeted proteomics via selected reaction monitoring, we show that the gene encoding the proinflammatory cytokine IL-36γ is a novel direct target of Nrf2 in keratinocytes and hepatocytes in vitro and in vivo. As a consequence, upregulation of IL-36γ expression occurred upon genetic or pharmacological activation of Nrf2 in the epidermis and in the normal and regenerating liver. Functional in vitro studies demonstrate that IL-36γ directly stimulates proliferation of keratinocytes. In particular, it induces expression of keratinocyte mitogens in fibroblasts, suggesting that the Nrf2-IL-36γ axis promotes keratinocyte proliferation through a double paracrine loop. These results provide mechanistic insight into Nrf2 action in the control of inflammation and cell proliferation through regulation of a proinflammatory cytokine with a key function in various inflammatory diseases.

Matrix Metalloproteinase 10 Degradomics in Keratinocytes and Epidermal Tissue Identifies Bioactive Substrates With Pleiotropic Functions.

Matrix metalloproteinases (MMPs) are important players in skin homeostasis, wound repair, and in the pathogenesis of skin cancer. It is now well established that most of their functions are related to processing of bioactive proteins rather than components of the extracellular matrix (ECM). MMP10 is highly expressed in keratinocytes at the wound edge and at the invasive front of tumors, but hardly any non-ECM substrates have been identified and its function in tissue repair and carcinogenesis is unclear. To better understand the role of MMP10 in the epidermis, we employed multiplexed iTRAQ-based Terminal Amine Isotopic Labeling of Substrates (TAILS) and monitored MMP10-dependent proteolysis over time in secretomes from keratinocytes. Time-resolved abundance clustering of neo-N termini classified MMP10-dependent cleavage events by efficiency and refined the MMP10 cleavage site specificity by revealing a so far unknown preference for glutamate in the P1 position. Moreover, we identified and validated the integrin alpha 6 subunit, cysteine-rich angiogenic inducer 61 and dermokine as novel direct MMP10 substrates and provide evidence for MMP10-dependent but indirect processing of phosphatidylethanolamine-binding protein 1. Finally, we sampled the epidermal proteome and degradome in unprecedented depth and confirmed MMP10-dependent processing of dermokine in vivo by TAILS analysis of epidermis from transgenic mice that overexpress a constitutively active mutant of MMP10 in basal keratinocytes. The newly identified substrates are involved in cell adhesion, migration, proliferation, and/or differentiation, indicating a contribution of MMP10 to local modulation of these processes during wound healing and cancer development. Data are available via ProteomeXchange with identifier PXD002474.

Caspase-1 activity is required for UVB-induced apoptosis of human keratinocytes.

Caspase-1 has a crucial role in innate immunity as the protease activates the proinflammatory cytokine prointerleukin(IL)-1ß. Furthermore, caspase-1 induces pyroptosis, a lytic form of cell death that supports inflammation. Activation of caspase-1 occurs in multi-protein complexes termed inflammasomes, which assemble upon sensing of stress signals. In the skin and in skin-derived keratinocytes, UVB irradiation induces inflammasome-dependent IL-1 secretion and sunburn. Here we present evidence that caspase-1 and caspase-4 are required for UVB-induced apoptosis. In UVB-irradiated human primary keratinocytes, apoptosis occurs significantly later than inflammasome activation but depends on caspase-1 activity. However, it proceeds independently of inflammasome activation. By a proteomics approach, we identified the antiapoptotic Bap31 as a putative caspase-1 substrate. Caspase-1-dependent apoptosis is possibly a recent process in evolution as it was not detected in mice. These results suggest a protective role of caspase-1 in keratinocytes during UVB-induced skin cancer development through the induction of apoptosis.

Macrophage matrix metalloproteinase-12 dampens inflammation and neutrophil influx in arthritis.

Resolution of inflammation reduces pathological tissue destruction and restores tissue homeostasis. Here, we used a proteomic protease substrate discovery approach, terminal amine isotopic labeling of substrates (TAILS), to analyze the role of the macrophage-specific matrix metalloproteinase-12 (MMP12) in inflammation. In murine peritonitis, MMP12 inactivates antithrombin and activates prothrombin, prolonging the activated partial thromboplastin time. Furthermore, MMP12 inactivates complement C3 to reduce complement activation and inactivates the chemoattractant anaphylatoxins C3a and C5a, whereas iC3b and C3b opsonin cleavage increases phagocytosis. Loss of these anti-inflammatory activities in collagen-induced arthritis in Mmp12(-/-) mice leads to unresolved synovitis and extensive articular inflammation. Deep articular cartilage loss is associated with massive neutrophil infiltration and abnormal DNA neutrophil extracellular traps (NETs). The NETs are rich in fibrin and extracellular actin, which TAILS identified as MMP12 substrates. Thus, macrophage MMP12 in arthritis has multiple protective roles in countering neutrophil infiltration, clearing NETs, and dampening inflammatory pathways to prepare for the resolution of inflammation.

The substrate degradome of meprin metalloproteases reveals an unexpected proteolytic link between meprin ß and ADAM10.

The in vivo roles of meprin metalloproteases in pathophysiological conditions remain elusive. Substrates define protease roles. Therefore, to identify natural substrates for human meprin α and ß we employed TAILS (terminal amine isotopic labeling of substrates), a proteomics approach that enriches for N-terminal peptides of proteins and cleavage fragments. Of the 151 new extracellular substrates we identified, it was notable that ADAM10 (a disintegrin and metalloprotease domain-containing protein 10)-the constitutive α-secretase-is activated by meprin ß through cleavage of the propeptide. To validate this cleavage event, we expressed recombinant proADAM10 and after preincubation with meprin ß, this resulted in significantly elevated ADAM10 activity. Cellular expression in murine primary fibroblasts confirmed activation. Other novel substrates including extracellular matrix proteins, growth factors and inhibitors were validated by western analyses and enzyme activity assays with Edman sequencing confirming the exact cleavage sites identified by TAILS. Cleavages in vivo were confirmed by comparing wild-type and meprin(-/-) mice. Our finding of cystatin C, elafin and fetuin-A as substrates and natural inhibitors for meprins reveal new mechanisms in the regulation of protease activity important for understanding pathophysiological processes.

CLIPPER: an add-on to the Trans-Proteomic Pipeline for the automated analysis of TAILS N-terminomics data.

Data analysis in proteomics is complex and with the extra challenges involved in the interpretation of data from N-terminomics experiments, this can be daunting.Therefore, we have devised a rational pipeline of steps to approach N-terminomics data analysis in a statistically based and valid manner. We have automated these steps in CLIPPER, an add-on to the Trans-Proteomic Pipeline(TPP). Applying CLIPPER to the analysis of N- terminomics data generated by terminal amine isotopic labeling of substrates (TAILS) enables high confidence peptide to protein assignment, protein N-terminal characterization and annotation, and for protease analysis readily allows protease substrate discovery with high confidence.