Determination of human factor VIII (AHG-associated protein)-antigen in endothelial cells from Xenopus laevis (XTH cells)

Summary AHG-associated protein (AHG-a.p.), the antigen of the blood-clotting factor VIII complex, is a specific endothelial cell marker. Primary (p-XTH) and established (XTH-2) endothelial cells from the hearts of Xenopus laevis tadpoles were assayed for the presence of this marker by means of immunological cross-reaction (recognition of common antigenic sites) with antiserum against human AHG-a.p. Radial imtnunodiffusion and rocket immunoelectrophoresis proved to be insufficiently sensitive, whereas immunofluorescence and a newly evaluated ELISA technique gave positive results. The very high sensitivity of the ELISA (less than 1/240000 of the AHG-a.p. in 0.1 ml human standard plasma can be detected) and the removal of interfering proteins by gel filtration also revealed the presence of AHG-a.p. in the fetal calf serum used in the culture medium; earlier investigations into this subject by a one-step radioimmunoassay had reported negative results. Specially adapted XTH-2 cells were grown in a proteinand serum-free hydrolysate medium in order to demonstrate the presence of a Xenopus-derived antigen that was immunoreactive with the anti-human AHG-a.p.     

Medicinal Plants of the Washambaa (Tanzania): Documentation and Ethnopharmacological Evaluation

Abstract: Medicinal plants are an important local resource for the Washambaa of the Western Usambara Mountains in Tanzania. In this study the Washambaa medicinal plants are inventoried. It is based on ethnobotanical fieldwork carried out over 15 months. This study presents for the first time an analysis of medicinal plants used in Africa with a ranking of these taxa by the frequency of their reported use. A total of 328 taxa were collected and yielded 2260 individual use reports. The most popular species are Myrica salicifolia and Toddalia asiatica. Subsequently, the use reports were arranged into 9 groups of medicinal uses based on the types of illness treated. The F actor of I nformant C onsensus (F_IC) is used in order to evaluate the ethnobotanical importance of the plants. The largest number of plants and of use reports are in the group of gastrointestinal disorders. For the most commonly used taxa, an ethnopharmacological evaluation was performed. Studies to evaluate the Washambaa therapeutic claims as well as toxicological data are still lacking for many of the species. This study will form the basis for pharmacological and phytochemical research on selected Washambaa medicinal plants.     

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.Matrix metalloproteinases (MMPs)¹ are extracellular zinc-dependent endoproteinases that are highly expressed in tissues undergoing remodeling processes during development, in response to injury, or as a result of neoplastic transformation (1–3). MMP10, also known as stromelysin-2, gained particular interest in the skin, because of its specific and strong expression in wound edge keratinocytes as well as at the invasive front of epithelial tumors (4–6). Overexpression of a constitutively active MMP10 mutant in wound keratinocytes in mice led to scattering of these cells at the tip of the migrating wound epithelium, altered β1-integrin expression, reduced AKT phosphorylation and increased apoptosis (7). Lack of MMP10 in a lung infection model affected genes that are involved in the regulation of apoptosis, cell proliferation, immune response and signal transduction (8). In the gut, bone marrow-derived MMP10 had a protective role in experimental colitis with implications in macrophage polarization (9). MMP10 released from hepatocytes and macrophages positively contributed to liver regeneration (10), whereby it promoted hepatocarcinogenesis in a complicated crosstalk with chemokine signaling (11). Most recently, Rohani et al. demonstrated a role for macrophage-derived MMP10 in moderating scar formation by controlling collagenase activity of dermal macrophages (12).Similar complex phenotypes have been associated with activities of most MMPs that, however, were not related to processing of extracellular matrix (ECM) proteins, the classical MMP substrates (13), but of bioactive mediators, including cell surface receptors, growth factor binding proteins, proteases, inhibitors, cytokines, and chemokines (14, 15). This changed the view on MMPs as simple tissue degraders to precise modulators of diverse processes, such as cell proliferation, migration, differentiation, angiogenesis, apoptosis and immune response (2, 16). As an example, functions of MMP3, the closest homolog of MMP10, in keratinocyte differentiation (17), tumor cell invasion (18), and immune cell recruitment (19) could be explained by processing of non-ECM proteins that have been identified as direct substrates of this protease (2, 20) in addition to ECM components (21). However, because MMP10 has been mostly neglected in the quest for new MMP substrates, it remains to be elucidated, if it also exerts its functions in part by processing of bioactive proteins whose identification is instrumental in understanding the mechanisms of action of MMP10 in tissue repair and carcinogenesis.Recently, we applied iTRAQ-based Terminal Amine Isotopic Labeling of Substrates (TAILS), a multiplexed quantitative proteomics workflow for identification of protease substrates in complex proteomes (22–24), to reveal new targets of MMP10 in secretomes from mouse embryonic fibroblasts (25). Moreover, to mimic MMP10 activity at the epidermal–dermal interface, we devised a new workflow that allowed monitoring both cellular origins and cleavages of substrates in mixed secretomes from keratinocytes and fibroblasts (26). However, this study focused on basement membrane components and missed additional information on cleavage kinetics. Thus, in this work, we employed time-resolved TAILS to identify novel MMP10 substrates in keratinocyte secretomes and mouse epidermal tissue, aiming at further characterizing the MMP10 substrate degradome in epidermal keratinocytes for a better understanding of its biological roles in the skin. Here, we identified novel bioactive substrates of MMP 10 in vitro and in the skin in vivo, which provide insight into its functions in wound repair and carcinogenesis. In addition, we revealed an unexpected preference of MMP10 for substrates that harbor a glutamate residue in P1 position, which might be exploited for the development of specific activity-based probes or inhibitors for this important wound- and tumor-related protease.     

Wound degradomics - current status and future perspectives

Proteases are pivotal modulators of extracellular matrix components and bioactive proteins at all phases of cutaneous wound healing and thereby essentially contribute to the successful reestablishment of skin integrity upon injury. As a consequence, disturbance of proteolytic activity at the wound site is a major factor in the pathology of chronic wounds. A large body of data acquired in many years of research provide a good understanding of how individual proteases may influence the repair process. The next challenge will be to integrate these findings and to elucidate the complex interactions of proteolytic enzymes, their inhibitors and substrates on a system-wide level. Here, we present novel approaches that might help to achieve this ambitious goal in cutaneous wound healing research.     

Time-resolved Analysis of the Matrix Metalloproteinase 10 Substrate Degradome

Proteolysis is an irreversible post-translational modification that affects intra- and intercellular communication by modulating the activity of bioactive mediators. Key to understanding protease function is the system-wide identification of cleavage events and their dynamics in physiological contexts. Despite recent advances in mass spectrometry-based proteomics for high-throughput substrate screening, current approaches suffer from high false positive rates and only capture single states of protease activity. Here, we present a workflow based on multiplexed terminal amine isotopic labeling of substrates for time-resolved substrate degradomics in complex proteomes. This approach significantly enhances confidence in substrate identification and categorizes cleavage events by specificity and structural accessibility of the cleavage site. We demonstrate concomitant quantification of cleavage site spanning peptides and neo-N and/or neo-C termini to estimate relative ratios of noncleaved and cleaved forms of substrate proteins. By applying this strategy to dissect the matrix metalloproteinase 10 (MMP10) substrate degradome in fibroblast secretomes, we identified the extracellular matrix protein ADAMTS-like protein 1 (ADAMTSL1) as a direct MMP10 substrate and revealed MMP10-dependent ectodomain shedding of platelet-derived growth factor receptor alpha (PDGFRα) as well as sequential processing of type I collagen. The data have been deposited to the ProteomeXchange Consortium with identifier PXD000503.Historically regarded as a mechanism for unspecific degradation of proteins, proteolysis is now recognized as a specific irreversible post-translational modification that affects major intra- and intercellular signaling processes (1, 2). Proteases specifically process bioactive proteins, their receptors, and associated proteins in an interconnected interaction network termed the protease web (3). Dysregulation of the protease web might cause or result from pathologies, such as impaired tissue repair, cancer and neurodegenerative diseases. Therefore, a better understanding of the functions of individual proteases and their interconnections within proteolytic networks is a prerequisite for exploiting proteases as targets for therapeutic intervention (4).To address this issue, several powerful technologies have been developed for the system-wide discovery of protease substrates, i.e. substrate degradomes, in complex and active proteomes (5, 6). A common principle of these mass spectrometry-based methods is the enrichment and monitoring of N-terminal peptides (protein neo-N termini) that are newly generated by a test protease (7). Protein N termini are enriched from complex proteomes either by chemical tagging and affinity resins (positive selection) or by depletion of internal peptides (negative selection) (7). Both principles have been successfully applied in various studies to characterize N-terminomes and to identify protease substrates using in vitro or cell-based systems and more recently also in vivo (8, 9). Negative enrichment approaches were further extended to the analysis of protein C termini (10, 11) and have the general advantage of recording data on naturally blocked (e.g. acetylated) N termini and internal peptides in the same experiment (8).Even if successful in identifying novel proteolytic cleavage events, which could also be validated by orthogonal methods, high-throughput substrate discovery approaches potentially suffer from high numbers of false positive identifications, particularly when employing in vitro systems (12). These have been reduced by monitoring abundances of N-terminal peptides at multiple time points after incubation of a proteome with a test protease (12). In this SILAC-based approach the authors efficiently distinguished critical from bystander cleavages, but it was limited to three time points. Therefore, it did not allow recording kinetic profiles of the relative abundance of N-terminal peptides that are required for determination of apparent kinetic parameters for processing events. Agard et al. elegantly overcame this limitation by use of selected reaction monitoring (SRM)¹ in combination with a positive N-terminal enrichment platform and determined apparent catalytic efficiencies for hundreds of caspase cleavage events in parallel (13). In a similar approach the same group characterized cellular responses to pro-apoptotic cancer drugs by recording time-courses for caspase-generated neo-N termini (14). Although very powerful and highly accurate in quantification, this method strongly exploited the canonical cleavage specificity of caspases after aspartate residues and required a two-stage process involving two types of mass spectrometers. Hence, it would be desirable to monitor the time-resolved generation of neo-N termini in complex proteomes in a single experiment by a simple and robust workflow in an unbiased manner.The development of such an analysis platform would require a reliable method for the system-wide characterization of protein N termini that is easy to perform, fast and highly multiplexible. All these criteria are met by iTRAQ-terminal amine isotopic labeling of substrates (TAILS), a multiplex N-terminome analysis technique that has been applied in 2plex and 4plex experiments to map the matrix metalloproteinase (MMP) 2 and MMP9 substrate degradomes in vitro (15) and most recently to quantitatively analyze the proteome and N-terminome of inflamed mouse skin in the presence or absence of the immune-modulatory protease MMP2 in vivo (8).Here, we exploited the multiplex capabilities of iTRAQ-TAILS by use of 8plex-iTRAQ reagents to monitor the generation of neo-N-terminal peptides by a test protease in complex samples over time. First, using GluC as a test protease with canonical cleavage specificity, we established a workflow for time-resolved substrate degradomics. Recording kinetic profiles significantly increased the confidence in identified cleavage events compared with binary systems and categorized primary cleavage specificities as well as secondary structure elements based on clusters of processing events with different efficiencies. By including data from before N-terminal enrichment, we extended our analysis to neo-C-terminal peptides and concomitantly monitored the generation of neo-N termini and neo-C termini as well as the decrease in abundance of the tryptic peptides spanning the cleavage sites in the same experiment. Next, we applied this approach to the time-resolved analysis of the hardly elucidated substrate degradome of matrix metalloproteinase 10 (MMP10). This important wound- and tumor-related protease is secreted by proliferating and migrating keratinocytes at the wound edge in close proximity to dermal fibroblasts and is also highly expressed in aggressive tumor cells (16–18). Our analysis revealed MMP10-dependent shedding of the platelet-derived growth factor receptor alpha (PDGFRα), processing of ADAMTS-like protein 1 (ADAMTSL1) and multiple cleavages of type I collagen, which could be validated and classified by time-resolved abundance profiles of their corresponding neo-N termini.     

In Vivo Assessment of Protease Dynamics in Cutaneous Wound Healing by Degradomics Analysis of Porcine Wound Exudates

Proteases control complex tissue responses by modulating inflammation, cell proliferation and migration, and matrix remodeling. All these processes are orchestrated in cutaneous wound healing to restore the skin''s barrier function upon injury. Altered protease activity has been implicated in the pathogenesis of healing impairments, and proteases are important targets in diagnosis and therapy of this pathology. Global assessment of proteolysis at critical turning points after injury will define crucial events in acute healing that might be disturbed in healing disorders. As optimal biospecimens, wound exudates contain an ideal proteome to detect extracellular proteolytic events, are noninvasively accessible, and can be collected at multiple time points along the healing process from the same wound in the clinics. In this study, we applied multiplexed Terminal Amine Isotopic Labeling of Substrates (TAILS) to globally assess proteolysis in early phases of cutaneous wound healing. By quantitative analysis of proteins and protein N termini in wound fluids from a clinically relevant pig wound model, we identified more than 650 proteins and discerned major healing phases through distinctive abundance clustering of markers of inflammation, granulation tissue formation, and re-epithelialization. TAILS revealed a high degree of proteolysis at all time points after injury by detecting almost 1300 N-terminal peptides in ∼450 proteins. Quantitative positional proteomics mapped pivotal interdependent processing events in the blood coagulation and complement cascades, temporally discerned clotting and fibrinolysis during the healing process, and detected processing of complement C3 at distinct time points after wounding and by different proteases. Exploiting data on primary cleavage specificities, we related candidate proteases to cleavage events and revealed processing of the integrin adapter protein kindlin-3 by caspase-3, generating new hypotheses for protease-substrate relations in the healing skin wound in vivo. The data have been deposited to the ProteomeXchange Consortium with identifier PXD001198.Proteases play pivotal roles in complicated tissue processes by influencing immune responses, epithelial and mesenchymal cell integrity, proliferation and migration, as well as extracellular matrix maturation and remodeling. As a prime example, they control all phases of cutaneous wound healing by participating in coagulation, complement activation, recruitment of immune cells, migration of keratinocytes and fibroblasts, angiogenesis, and formation of the scar tissue (1, 2). Immediately after injury a blood clot is formed through a series of interconnected proteolytic processing events of coagulation factors to initially seal the site of damage and to provide a provisional fibrin matrix (3, 4). Soon after and interfacing with coagulation, the complement system is activated to fight invading bacteria. During the inflammatory phase (day 1 to 3) the kallikrein–kinin axis controls vasodilation and vascular permeability, and leukocytes enter the wounded tissue in response to pro-inflammatory chemo-attractants whose activity is regulated by limited proteolysis (5). Upon activation of the plasmin system, the fibrin clot is proteolytically degraded to facilitate migration of keratinocytes from the epidermis and the hair follicles as well as of macrophages and fibroblasts in the granulation tissue. These migratory events that occur during the phase of new tissue formation (days 3–10) are further promoted by matrix metalloproteinases (MMPs)¹ that are activated by plasmin and concomitantly modulate tissue influx of immune cells and resolution of inflammation (6, 7). MMPs are also heavily involved in matrix remodeling and scar formation as the final step of skin repair that starts 1 to 2 weeks after injury, but may continue for up to 1 year or more (8).As a consequence of their crucial roles in the healing skin, wound proteases have been implicated in the pathogenesis of healing disorders (9). Impaired wound healing has detrimental consequences and often leads to the development of chronic, nonhealing ulcers. In particular, patients suffering from vascular disease, diabetes, or autoimmune disorders frequently develop chronic skin ulcers. Chronic wounds have become a major problem in industrialized western countries with their rising rates of obesity and the increasing life expectancy, putting also an enormous burden on health systems (10). Because MMPs received much attention in chronic wound repair (11, 12), current diagnostic tests rely on assessment of general MMP activity in wound swabs (13), but suffer from lack of specificity and fail in many cases in predicting the actual wound status. Hence, novel multiparameter point-of-care tests are needed that integrate multiple proteolytic events to deliver robust results on aberrant proteolysis as an indicator for chronic wound progression (14).Proteolytic cleavages in major cascades, such as blood coagulation and complement activation, have been mapped in great detail through seminal biochemical studies (15, 16). In vitro studies used purified or recombinant proteins or monitored processing of radioactively labeled components spiked into activated blood plasma (17, 18). Later, the invention of monoclonal antibodies and/or active site labels also enabled the analysis of endogenous proteolytically activated coagulation factors and complement components in in vivo samples (19). However, none of these techniques allowed directly recording the actual interconnected cleavage events of these complex proteolytic activation cascades in vivo and in response to a natural incidence like tissue injury, a prerequisite to better understand their disturbances in pathology. Addressing this limitation, mass spectrometry-based degradomics technologies have been developed that identify and relatively quantify protein N termini in complex biological samples (20–22). One of these methods, Terminal Amine Isotopic Labeling of Substrates (TAILS), was successfully applied in vitro to identify novel substrates of individual proteases (23–28) and more recently also in vivo to systematically assess protease activity in complex tissue samples (29, 30). TAILS has unique multiplexing capabilities and thus is particularly suited for analyzing the N-terminome at multiple time points after the stimulus (31) as required for the time-resolved analysis of proteolytic events at critical turning points after skin injury (32).An optimal sample for the system-wide analysis of protease activity in cutaneous wound healing should be easily and preferentially noninvasively accessible, cover most cleavage events, and be ideally obtained from the same wound at multiple time points after wounding. This is the case for wound exudates, which can be either directly collected from the wound site (33, 34) or extracted from wound dressings (35). Several proteomic analyses of wound fluids have been performed that mostly focused on the quantitative comparison of proteins in fluids from normal and impaired healing (33, 35). The most recent studies covered a significant proportion of the wound proteome and recorded differential protein abundances at single states of chronic manifestation or normal healing (35). However, these analyses did not integrate data on healing progression and/or functional modifications to the wound proteome along the healing process. Importantly, several studies suggest a higher predictive power of post-translational modifications than relative protein abundances for disease progression (36, 37). Hence, recording proteolytic signatures at critical time points after wounding is a promising approach to define pivotal events in acute healing that might be disturbed in healing impairments.Here, we exploited the power of multiplexed iTRAQ-TAILS to globally analyze the wound fluid proteome and N-terminome at multiple time points after injury. We identified more than 650 proteins and almost 1300 protein N termini from exudates collected in a clinically relevant pig wound healing model. By combining quantitative proteome and N-terminome analyses, we temporally discerned major phases of acute wound healing and mapped key cleavages in blood coagulation and complement activation. Further, we revealed protease dynamics through identification, quantification, and relative weighting of multiple cleavages in complement C3. Finally, by integrating data on known cleavage site specificities we related groups of proteases to identified cleavage sites and established direct cleavage of the integrin adapter protein kindlin-3 by caspase-3, which might play an important role in immune cell apoptosis during cutaneous wound healing.     

In vitro degradation and antitumor activity of oxime bond-linked daunorubicin-GnRH-III bioconjugates and DNA-binding properties of daunorubicin-amino acid metabolites

Bioconjugates with receptor-mediated tumor-targeting functions and carrying cytotoxic agents should enable the specific delivery of chemotherapeutics to malignant tissues, thus increasing their local efficacy while limiting the peripheral toxicity. In the present study, gonadotropin-releasing hormone III (GnRH-III; Glp-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH₂) was employed as a targeting moiety to which daunorubicin was attached via oxime bond, either directly or by insertion of a GFLG or YRRL tetrapeptide spacer. The in vitro antitumor activity of the bioconjugates was determined on MCF-7 human breast and HT-29 human colon cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Their degradation/stability (1) in human serum, (2) in the presence of cathepsin B and (3) in rat liver lysosomal homogenate was analyzed by liquid chromatography in combination with mass spectrometry. The results show that (1) all synthesized bioconjugates have in vitro antitumor effect, (2) they are stable in human serum at least for 24 h, except for the compound containing an YRRL spacer and (3) they are hydrolyzed by cathepsin B and in the lysosomal homogenate. To investigate the relationship between the in vitro antitumor activity and the structure of the bioconjugates, the smallest metabolites produced in the lysosomal homogenate were synthesized and their binding to DNA was assessed by fluorescence spectroscopy. Our data indicate that the incorporation of a peptide spacer in the structure of oxime bond-linked daunorubicin–GnRH-III bioconjugates is not required for their antitumor activity. Moreover, the antitumor activity is influenced by the structure of the metabolites (daunorubicin–amino acid derivatives) and their DNA-binding properties.     

The extracellular matrix (mesoglea) of hydrozoan jellyfish and its ability to support cell adhesion and spreading

The outer mesoglea (extracellular matrix; ECM) of hydrozoan jellyfish was found to contain a species-specific meshwork of striated fibers of different diameters. In the mesoglea, molecules were identified which exhibit several features of well known vertebrate ECM: a laminin-like molecule which appears cross-shaped on electronmicrographs, a fibronectin-like molecule (both detectable by their immunoreactivity at the exumbrella side) and a species-specific collagen consisting of 3 different -chains of which at least 2 can be decorated with con A. The -chains are linked by disulfide bridges. Acetic acid extraction of the mesoglea and subsequent salt precipitation yields fibrils which appear banded in the electron microscope and support species-specific adhesion and spreading of isolated tissue. These precipitated fibrils are mainly composed of the disulfide-linked collagen.     

Iota-carrageenan extracted from red algae is a potent inhibitor of SARS‐CoV-2 infection in reconstituted human airway epithelia

Iota-carrageenan (IC) nasal spray, a medical device approved for treating respiratory viral infections, has previously been shown to inhibit the ability of a variety of respiratory viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), to enter and replicate in the cell by interfering with the virus binding to the cell surface. The aim of this study was to further investigate the efficacy and safety of IC in SARS-CoV-2 infection in advanced in vitro models of the human respiratory epithelium, the primary target and entry port for SARS-CoV-2. We extended the in vitro safety assessment of nebulized IC in a 3-dimensional model of reconstituted human bronchial epithelium, and we demonstrated the efficacy of IC in protecting reconstituted nasal epithelium against viral infection and replication of a patient-derived SARS-CoV-2 strain. The results obtained from these two advanced models of human respiratory tract epithelia confirm previous findings from in vitro SARS-CoV-2 infection assays and demonstrate that topically applied IC can effectively prevent SARS-CoV-2 infection and replication. Moreover, the absence of toxicity and functional and structural impairment of the mucociliary epithelium demonstrates that the nebulized IC is well tolerated.