Crosstalk between Platelets and SARS-CoV-2: Implications in Thrombo-Inflammatory Complications in COVID-19.

The SARS-CoV-2 virus, causing the devastating COVID-19 pandemic, has been reported to affect platelets and cause increased thrombotic events, hinting at the possible bidirectional interactions between platelets and the virus. In this review, we discuss the potential mechanisms underlying the increased thrombotic events as well as altered platelet count and activity in COVID-19. Inspired by existing knowledge on platelet-pathogen interactions, we propose several potential antiviral strategies that platelets might undertake to combat SARS-CoV-2, including their abilities to internalize the virus, release bioactive molecules to interfere with viral infection, and modulate the functions of immune cells. Moreover, we discuss current and potential platelet-targeted therapeutic strategies in controlling COVID-19, including antiplatelet drugs, anticoagulants, and inflammation-targeting treatments. These strategies have shown promise in clinical settings to alleviate the severity of thrombo-inflammatory complications and reduce the mortality rate among COVID-19 patients. In conclusion, an in-depth understanding of platelet-SARS-CoV-2 interactions may uncover novel mechanisms underlying severe COVID-19 complications and could provide new therapeutic avenues for managing this disease.

Citrus tangerine pith extract alleviates hypoxia-induced ileum damage in mice by modulating intestinal microbiota.

Visitors to high altitude are susceptible to hypoxia-induced acute intestinal mucosal barrier injury and severe gastrointestinal disorders, which are life-threatening. Citrus tangerine pith extract (CTPE) is rich in pectin and flavonoids and has been proved to enhance intestinal health and improve gut dysbiosis. In this study, we aim to explore the protective effect of CTPE on ileum injury induced by intermittent hypobaric hypoxia in a mouse model. Balb/c mice were divided into blank normoxia (BN), blank hypobaric hypoxia (BH), hypobaric hypoxia plus CTPE (TH), and hypobaric hypoxia plus Rhodiola extract (RH) groups. From the 6th day of gavage, mice in BH, TH, and RH groups were transferred into a hypobaric chamber at a simulated elevation of 6000 m for 8 hours per day for 10 days. Then half the mice were tested for small intestine movement, and others were used to evaluate intestinal physical barrier function, inflammation, and gut microbiota. Results showed that CTPE reversed the increase of intestinal peristalsis, effectively attenuated impaired structural integrity of ileum, improved the mRNA and protein expression levels of tight junction proteins, and reduced serum D-LA content in mice to alleviate hypoxia-induced mucosal barrier damage. Moreover, CTPE supplementation ameliorated hypoxia-induced intestinal inflammation response by significantly downregulating the proinflammatory cytokines IL-6, TNF-α and IFN-γ. By 16S rDNA gene sequencing of gut microbiota, CTPE significantly increased the abundance of probiotic Lactobacillus, suggesting that CTPE may be used as a prebiotic to regulate ecology of intestinal microorganisms. In addition, Spearman rank correlation analysis revealed that changed gut microbiota were significantly correlated with alteration of intestinal barrier function indexes. Taken together, these results indicate that CTPE effectively alleviates hypoxia-induced intestinal injury in mice and enhances intestinal integrity and barrier function by altering intestinal microbiota composition.

[Enteral nutrition in critically ill patients: routine monitoring gastric residual volume or not].

Gastric residual volume (GRV) has been widely used in clinical practice for a long time as an essential indicator of gastrointestinal motility and enteral nutrition tolerance. Recent studies have questioned the rationality of monitoring GRV routinely in critically ill patients. A "one size fits all" principle is not an optimal solution in the clinical practice of enteral nutrition in critically ill patients because of the huge heterogeneity of the intensive care unit (ICU) population. Each method of monitoring GRV has advantages and disadvantages. With the widespread clinical application of harmless monitoring technologies such as ultrasound, the gastric suctioning method maybe used less and less. The management of enteral nutrition focuses on identifying the factors and triggers of gastric retention, attempting to solve them from the perspective of prevention and elimination of high-risk factors for aspiration and analysis of pathophysiological mechanisms, eliminating all negative hindering factors, and actively promoting the concept of "creating conditions for enteral nutrition". As a critical nutrition management team, it is necessary to pay attention to the continuous improvement of enteral nutrition management culture and rationally use diversified management strategies, which may be more important than monitoring GRV.

Gut dysbiosis induced by antibiotics is improved by tangerine pith extract in mice.

The gut microbiota can affect the intestinal barrier and other physiological functions. Tangerine pith contains various citrus pectins, which modulate the gut microbiota in a structure-dependent manner. We hypothesized that tangerine pith extract (TPE) has prebiotic effects on gut dysbiosis in antibiotic-treated mice. TPE was prepared via acid hydrolysis and isolated using an ultrafiltration method. Gut dysbiosis was induced through treatment with antibiotic cocktail for 7 days in mice. After spontaneous recovery or being supplemented with 0.5 mL TPE for 14 days, the gut microbiota composition and other parameters were examined. The 16s ribosomal DNA high-throughput sequencing and analysis confirmed that TPE positively improved the gut microbiota. The relative abundance of some probiotics such as [Ruminococcus]_torques_group, [Clostridium]_innocuum_group, and Enterorhabdus were significantly increased and harmful bacteria such as Alistipes were decreased in TPE-supplemented mice. Furthermore, the production of propionate and butyrate in the colonic contents were significantly enhanced in the TPE-treated mice. Serum D-lactic acid and diamine oxidase activity were increased in the antibiotic treatment group and reversed in the TPE-treated group. Moreover, protein expression of intestinal occludin was significantly increased by TPE. In conclusion, TPE is effective in restoring the gut microbiota in mice after antibiotic treatment and may act as a prebiotic in combatting gut dysbiosis.

Role of mitochondrial reactive oxygen species in homeostasis regulation.

Mitochondria are the main source of reactive oxygen species (ROS) in cells. Early studies have shown that mitochondrial reactive oxygen species (mROS) are related to the occurrence and adverse outcomes of many diseases, and are thus regarded as an important risk factor that threaten human health. Recently, increasing evidence has shown that mROS are very important for an organism's homeostasis. mROS can regulate a variety of signaling pathways and activate the adaptation and protection behaviors of an organism under stress. In addition, mROS also regulate important physiological processes, such as cell proliferation, differentiation, aging, and apoptosis. Herein, we review the mechanisms of production, transformation, and clearance of mROS and their biological roles in different physiological processes.

Expression and significance of SOX B1 genes in glioblastoma multiforme patients.

The overall survival of glioblastoma multiforme (GBM) patients remains poor. To improve patient outcomes, effective diagnostic and prognostic biomarkers for GBM are needed. In this study, we first applied bioinformatic analyses to identify biomarkers for GBM, focusing on SOX (sex-determining region on the Y chromosome (SRY)-related high mobility group (HMG) box) B1 family members. The ONCOMINE, GEPIA, LinkedOmics and CCLE databases were used to assess mRNA expression levels of the SOX B1 family members in different cancers and normal tissue. Further bioinformatic analysis was performed using the ONCOMINE database in combination with the LinkedOmics data set to identify the prognostic value of SOX B1 family members for GBM. We found mRNA expression levels of all tested SOX B1 genes were significantly increased in GBM. In the LinkedOmics database, increased expression of SOX3 indicated a better overall survival. In GEPIA databases, increased expression of all SOX B1 family members suggested an improved overall survival, but none of them were statistically different. Then, Transwell assays and wound healing were employed to evaluate the motility and invasive captivity of U251 cells when silencing SOX2 and SOX3. We found exogenous inhibition of SOX2 appeared to reduce the migration and invasion of U251 cells in vitro. Collectively, our research suggested that SOX2 might serve as a cancer-promoting gene to identify high-risk GBM patients, and SOX3 had the potential to be a prognostic biomarker for GBM patients.

High Fat-High Fructose Diet-Induced Changes in the Gut Microbiota Associated with Dyslipidemia in Syrian Hamsters.

Aim: The objective of this study was to characterize the early effects of high fructose diets (with and without high fat) on both the composition of the gut microbiota and lipid metabolism in Syrian hamsters, a reproducible preclinical model of diet-induced dyslipidemia. Methods: Eight-week-old male hamsters were fed diets consisting of high-fat/high-fructose, low-fat/high-fructose or a standard chow diet for 14 days. Stool was collected at baseline (day 0), day 7 and day 14. Fasting levels of plasma triglycerides and cholesterol were monitored on day 0, day 7 and day 14, and nonfasting levels were also assayed on day 15. Then, 16S rRNA sequencing of stool samples was used to determine gut microbial composition, and predictive metagenomics was performed to evaluate dietary-induced shifts in deduced microbial functions. Results: Both high-fructose diets resulted in divergent gut microbiota composition. A high-fat/high-fructose diet induced the largest shift in overall gut microbial composition, with dramatic shifts in the Firmicute/Bacteroidetes ratio, and changes in beta diversity after just seven days of dietary intervention. Significant associations between genus level taxa and dietary intervention were identified, including an association with Ruminococceace NK4A214 group in high-fat/high-fructose fed animals and an association with Butryimonas with the low-fat/high-fructose diet. High-fat/high-fructose feeding induced dyslipidemia with increases in plasma triglycerides and cholesterol, and hepatomegaly. Dietary-induced changes in several genus level taxa significantly correlated with lipid levels over the two-week period. Differences in microbial metabolic pathways between high-fat/high-fructose and low-fat/high-fructose diet fed hamsters were identified, and several of these pathways also correlated with lipid profiles in hamsters. Conclusions: The high-fat/high-fructose diet caused shifts in the host gut microbiota. These dietary-induced alterations in gut microbial composition were linked to changes in the production of secondary metabolites, which contributed to the development of metabolic syndrome in the host.

Role of Gut Microbiota in Neuroendocrine Regulation of Carbohydrate and Lipid Metabolism via the Microbiota-Gut-Brain-Liver Axis.

Gut microbiota play an important role in maintaining intestinal health and are involved in the metabolism of carbohydrates, lipids, and amino acids. Recent studies have shown that the central nervous system (CNS) and enteric nervous system (ENS) can interact with gut microbiota to regulate nutrient metabolism. The vagal nerve system communicates between the CNS and ENS to control gastrointestinal tract functions and feeding behavior. Vagal afferent neurons also express receptors for gut peptides that are secreted from enteroendocrine cells (EECs), such as cholecystokinin (CCK), ghrelin, leptin, peptide tyrosine tyrosine (PYY), glucagon-like peptide-1 (GLP-1), and 5-hydroxytryptamine (5-HT; serotonin). Gut microbiota can regulate levels of these gut peptides to influence the vagal afferent pathway and thus regulate intestinal metabolism via the microbiota-gut-brain axis. In addition, bile acids, short-chain fatty acids (SCFAs), trimethylamine-N-oxide (TMAO), and Immunoglobulin A (IgA) can also exert metabolic control through the microbiota-gut-liver axis. This review is mainly focused on the role of gut microbiota in neuroendocrine regulation of nutrient metabolism via the microbiota-gut-brain-liver axis.

The Role of the Gut Microbiota in Lipid and Lipoprotein Metabolism.

Both environmental and genetic factors contribute to relative species abundance and metabolic characteristics of the intestinal microbiota. The intestinal microbiota and accompanying microbial metabolites differ substantially in those who are obese or have other metabolic disorders. Accumulating evidence from germ-free mice and antibiotic-treated animal models suggests that altered intestinal gut microbiota contributes significantly to metabolic disorders involving impaired glucose and lipid metabolism. This review will summarize recent findings on potential mechanisms by which the microbiota affects intestinal lipid and lipoprotein metabolism including microbiota dependent changes in bile acid metabolism which affects bile acid signaling by bile acid receptors FXR and TGR5. Microbiota changes also involve altered short chain fatty acid signaling and influence enteroendocrine cell function including GLP-1/GLP-2-producing L-cells which regulate postprandial lipid metabolism.

Screening of chemical libraries in pursuit of kallikrein-5 specific inhibitors for the treatment of inflammatory dermatoses.

Background Aberrant kallikrein activity is observed in a number of inflammatory dermatoses. Up-regulation of kallikrein-5 (KLK5) activity leads to uncontrolled skin desquamation and cleavage of proteinase-activated receptor-2 (PAR2), causing the release of pro-inflammatory cytokines and disruption of epidermal barrier function. This study aimed to identify KLK5-specific small molecule inhibitors which can serve as the foundation of a novel therapeutic for inflammatory skin disorders. Methods Five chemical libraries (13,569 compounds total) were screened against recombinant KLK5 using a fluorogenic enzymatic assay. Secondary validation was performed on the top 22 primary hits. All hits were docked in the KLK5 crystal structure to rationalize their potential interactions with the protein. Results A naturally occurring compound derived from the wood of Caesalpinia sappan (Brazilin) was identified as a novel KLK5 inhibitor (IC50: 20 µM, Ki: 6.4 µM). Docking suggests that the phenolic moiety of Brazilin binds in the S1-pocket of KLK5 and forms a H-bond with S195 side chain. KLK14 was also found to be susceptible to inhibition by Brazilin with a calculated IC50 value of 14.6 µM. Conclusions Natural KLK5 small molecule inhibitors such as Brazilin, are ideal for topical skin disease drug design and remain a promising therapeutic for severe cases of inflammatory skin disorders. Optimized KLK inhibitors may have increased efficacy as therapeutics and warrant further investigation.

Aberrant endoplasmic reticulum stress mediates coronary artery spasm through regulating MLCK/MLC2 pathway.

Coronary artery spasm (CAS) is a pathophysiological phenomenon that may cause myocardial infarction and lead to circulatory collapse and death. Aberrant endoplasmic reticulum (ER) stress causes accumulation of misfolding proteins and has been reported to be involved in a variety of vascular diseases. The present study investigated the role of ER stress in the development of CAS and explored the possible molecular mechanisms. Initially, it was found that ER stress markers were elevated in response to drug-induced vascular smooth muscle cells (VSMCs) contraction. Pharmacologic activation of ER stress using Tunicamycin (Tm) persistently induced CAS and significantly promoted Pituitrin-induced CAS in mice as well as in a collagen gel contraction assay. On the contrary, pharmacologic inhibition of ER stress using 4-phenylacetic acid (4-PBA) completely blunted Pituitrin-induced CAS development in mice. Moreover, during the drug-induced VSMCs contraction, expression of ER stress markers were increased in parallel to those of myosin light chain kinase (MLCK) and phosphor-MLC2 (p-MLC2, at Ser19). After inhibiting MLCK activity by using its specific inhibitor ML-7, the ER stress activator Tm failed to activate the MLCK/MLC2 pathway and could neither trigger CAS in mice nor induce VSMCs contraction in vitro. Our results suggested that aberrant ER stress mediated CAS via regulating the MLCK/MLC2 pathway. ER stress activators might be more robust than the common drugs (Pituitrin or acetylcholine) as to induce vasocontraction and thus may serve as potential therapeutics against chronic bleeding, while its inhibitor might be useful for treatment of severe CAS caused by other medication.

Functional proteomic profiling reveals KLK13 and TMPRSS11D as active proteases in the lower female reproductive tract.

Background: Cervical-vaginal fluid (CVF) hydrates the mucosa of the lower female reproductive tract and is known to contain numerous proteases. The low pH of CVF (4.5 or below in healthy women of reproductive age) is a uniquely human attribute and poses a challenge for the proteolytic functioning of the proteases identified in this complex biological fluid. Despite the abundance of certain proteases in CVF, the proteolytic activity and function of proteases in CVF is not well characterized. Methods: In the present study, we employed fluorogenic substrate screening to investigate the influence of pH and inhibitory compounds on the proteolytic activity in CVF. Activity-based probe (ABP) proteomics has evolved as a powerful tool to investigate active proteases within complex proteomes and a trypsin-specific ABP was used to identify active proteases in CVF. Results: Serine proteases are among the most abundant proteins in the CVF proteome. Labeling human CVF samples with the trypsin-specific ABP revealed serine proteases transmembrane protein serine 11D and kallikrein-related peptidase 13 as active proteases in CVF. Furthermore, we demonstrated that the proteolytic activity in CVF is highly pH-dependent with an almost absolute inhibition of trypsin-like proteolytic activity at physiological pH levels. Conclusions: These findings provide a framework to understand proteolytic activity in CVF. Furthermore, the present results provide clues for a novel regulatory mechanism in which fluctuations in CVF pH have the potential to control the catalytic activity in the lower female reproductive tract.

Discovery of Antimicrobial Peptides in Cervical-Vaginal Fluid from Healthy Nonpregnant Women via an Integrated Proteome and Peptidome Analysis.

Cervical-vaginal fluid (CVF) covers the lower part of the female reproductive system and functions in the homeostasis and immunity of the surrounding tissues. In contrast to the CVF proteome of both nonpregnant and pregnant women, the CVF peptidome has not been reported to date. In the current study, we identified 1087 proteins in CVF, of which 801 proteins were not previously identified in CVF proteomes. The presence of the tissue-specific proteins oviductal glycoprotein 1 and tubulin polymerization-promoting protein family member 3 strongly suggests that the tissues of the upper female reproductive tract contribute to the protein composition of CVF. The tremendous catalytic potential of CVF was highlighted by the identification of 85 proteases and the detection of pH-dependent trypsin-like proteolytic activity. Over 1000 endogenous peptides were detected in the CVF peptidome, and 39 peptides are predicted to have antimicrobial activity. The detailed proteomic and peptidomic analysis of CVF will further aid in the delineation of pathways related to reproduction, immunity and host defense, and assist in developing new biomarkers for malignant and other diseases of the female reproductive tract. Data are available via ProteomeXchange with identifiers PXD004450 (CVF peptidome) and PDX004363 (CVF proteome).

Biochemical and functional characterization of the human tissue kallikrein 9.

Human tissue kallikrein 9 (KLK9) is a member of the kallikrein-related family of proteases. Despite its known expression profile, much less is known about the functional roles of this protease and its implications in normal physiology and disease. We present here the first data on the biochemical characterization of KLK9, investigate parameters that affect its enzymatic activity (such as inhibitors) and provide preliminary insights into its putative substrates. We show that mature KLK9 is a glycosylated chymotrypsin-like enzyme with strong preference for tyrosine over phenylalanine at the P1 cleavage position. The enzyme activity is enhanced by Mg2+ and Ca2+, but is reversibly attenuated by Zn2+ KLK9 is inhibited in vitro by many naturally occurring or synthetic protease inhibitors. Using a combination of degradomic and substrate specificity assays, we identified candidate KLK9 substrates in two different epithelial cell lines [the non-tumorigenic human keratinocyte cells (HaCaT) and the tumorigenic tongue squamous carcinoma cells (SCC9)]. Two potential KLK9 substrates [KLK10 and midkine (MDK)] were subjected to further validation. Taken together, our data delineate some functional and biochemical properties of KLK9 for future elucidation of the role of this enzyme in health and disease.

Proteomic and peptidomic analysis of human sweat with emphasis on proteolysis.

Sweat is produced by eccrine and apocrine glands and represents a biological fluid with established roles in thermo-regulation and host infection defense. The composition of sweat is highly dynamic and alters significantly in various skin and other disorders. Therefore, in-depth profiling of sweat protein composition is expected to augment our understanding of the pathobiology of several skin diseases and may lead to the identification of useful sweat-based disease biomarkers. We here reported an in-depth analysis of the human sweat proteome and peptidome. Sweat was collected from healthy males and healthy females of ages 20-60years, following strenuous exercise. Two sweat pools were prepared (1 for males and 1 for females) and were subjected to sample preparation for mass spectrometric analysis. We identified a total of 861 unique proteins during our proteomic analysis and 32,818 endogenous peptides (corresponding to additional 1067 proteins) from our peptidomics workflow. As expected, the human skin was identified as the most abundant source of sweat proteins and peptides. Several skin proteases and protease inhibitors were identified in human sweat, highlighting the intense proteolytic activity of human skin. The presence of several antimicrobial peptides supports the functional roles of sweat in host defense and innate immunity. SIGNIFICANCE: Sweat is a skin-associated biological fluid, secreted by eccrine and apocrine glands, with essential function in body thermo-regulation and host infection defense. In the present study, we performed in-depth profiling of both sweat proteome and peptidome composition. Our data provide the most in-depth characterization of the skin's catalytic network present in sweat. For the first time, we brought to light novel peptides in human sweat that potentially have antimicrobial activity, which highlight the important role of this fluid in innate immunity. All these findings allow us to have a better understanding of the complex web of proteases in skin and may act as a platform for the future discovery of novel skin biomarkers.

Association between Atrial Fibrillation and Three-Year Mortality in Nondiabetic Patients with Acute First-Ever Ischemic Stroke.

BACKGROUND: Diabetes mellitus (DM) is a risk factor for atrial fibrillation (AF) and is known to be an important risk factor for death from stroke. The influence of AF on long-term outcomes in patients with ischemic stroke remains controversial. To clarify the exact influence of AF on stroke outcome and exclude the effect from DM, we investigated the influence of AF on the 3-year outcomes of nondiabetic patients with acute first-ever ischemic stroke. METHODS: Five-hundred seventy-four nondiabetic patients with acute first-ever ischemic stroke were enrolled and had been followed for 3 years. Patients were divided into 2 groups according to whether AF was diagnosed or not. Clinical presentations, risk factors for stroke, laboratory data, comorbidities, and outcomes were recorded. RESULTS: A total of 107 patients (18.6%) had AF. The age was significantly older in patients with AF. Total anterior circulation syndrome occurred more frequently among patients with AF (P < .001). The mean length of stay in the acute ward was significantly higher in patients with AF (P < .001). Furthermore, dependent functional status following discharge was higher in patients with AF (P < .001). Multivariate Cox regression revealed that AF is a significant predictor of 3-year all-cause mortality (hazard ratio = 1.98, 95% confidence interval = 1.07-3.67, P = .022). CONCLUSIONS: AF is associated with increased risk of 3-year mortality in nondiabetic patients with acute first-ever ischemic stroke. Careful cardiac evaluation and treatment are essential in patients with AF and stroke.

Association between pneumonia in acute stroke stage and 3-year mortality in patients with acute first-ever ischemic stroke.

The influence of pneumonia in acute stroke stage on the clinical presentation and long-term outcomes of patients with acute ischemic stroke is still controversial. We investigate the influence of pneumonia in acute stroke stage on the 3-year outcomes of patients with acute first-ever ischemic stroke. Nine-hundred and thirty-four patients with acute first-ever ischemic stroke were enrolled and had been followed for 3years. Patients were divided into two groups according to whether pneumonia occurred during acute stroke stage or not. Clinical presentations, risk factors for stroke, laboratory data, co-morbidities, and outcomes were recorded. The result showed that a total of 100 patients (10.7%) had pneumonia in acute stroke stage. The prevalence of older age, atrial fibrillation was significantly higher in patients with pneumonia in acute stroke stage. Total anterior circulation syndrome and posterior circulation syndrome occurred more frequently among patients with pneumonia in acute stroke stage (P<0.001 and P=0.009, respectively). Multivariate Cox regression revealed that pneumonia in acute stroke stage is a significant predictor of 3-year mortality (hazard ratio=6.39, 95% confidence interval=4.03-10.11, P<0.001). In conclusion, pneumonia during the acute stroke stage is associated with increased risk of 3-year mortality. Interventions to prevent pneumonia in acute stroke stage might improve ischemic stroke outcome.

Association between renal dysfunction and 3-year mortality in patients with acute first-ever ischemic stroke.

OBJECTIVE: The influence of renal dysfunction on the clinical presentation and outcomes of patients with acute ischemic stroke is still controversial. We investigate the influence of renal dysfunction on the outcomes of patients with acute first-ever ischemic stroke. METHODS: Nine-hundred thirty-four patients with acute first-ever ischemic stroke were enrolled and followed for 3 years. Renal function was assessed using the equation of the Modification Diet for Renal Disease for estimated glomerular filtration rate (eGFR). Serum creatinine levels were obtained within 3 days of acute stroke onset. Reduced eGFR was defined as eGFR<60ml/min/1.73m(2). Clinical presentation, risk factors for stroke, laboratory data, co-morbidities, and outcomes were recorded. RESULTS: Total 264 patients (28.3%) had a reduced eGFR. The prevalence of older age, hypertension, and atrial fibrillation was significantly higher in patients with a reduced eGFR. Total anterior circulation syndrome occurred more frequently among patients with a reduced eGFR (P=0.010). Multivariate Cox regression revealed that a reduced eGFR is a significant predictor of 3-year mortality (HR=1.67, 95% CI=1.06-2.62, P=0.026). CONCLUSION: Reduced eGFR during the acute stroke stage is associated with increased risk of 3-year mortality. Furthermore, risk of acute complications and poor functional outcomes following discharge was significantly higher in patients with a reduced eGFR.

Novel Biological Substrates of Human Kallikrein 7 Identified through Degradomics.

Kallikrein-related peptidases (KLKs) are a group of serine proteases widely expressed in various tissues and involved in a wide range of physiological and pathological processes. Although our understanding of the pathophysiological roles of most KLKs has blossomed in recent years, identification of the direct endogenous substrates of human KLKs remains an unmet objective. In this study we employed a degradomics approach to systemically investigate the endogenous substrates of KLK7 in an effort to understand the molecular pathways underlying KLK7 action in skin. We identified several previously known as well as novel protein substrates. Our most promising candidates were further validated with the use of targeted quantitative proteomics (selected reaction monitoring methods) and in vitro recombinant protein digestion assays. Our study revealed midkine, CYR61, and tenascin-C as endogenous substrates for KLK7. Interestingly, some of these substrates (e.g. midkine) were prone to proteolytic cleavage only by KLK7 (and not by other skin-associated KLKs), whereas others (e.g. CYR61 and tenascin-C) could be digested by several KLKs. Furthermore, using melanoma cell line, we show that KLK7-mediated cleavage of midkine results in an overall reduction in the pro-proliferative and pro-migratory effect of midkine. An inverse relation between KLK7 and midkine is also observed in human melanoma tissues. In summary, our degradomics approach revealed three novel endogenous substrates for KLK7, which may shed more light on the pathobiological roles of KLK7 in human skin. Similar substrate screening approaches could be applied for the discovery of biological substrates of other protease.

Putative kallikrein substrates and their (patho)biological functions.

Human tissue kallikreins (KLKs) represent the largest contiguous group of protease genes within our genome. All 15 KLK genes co-localize within approximately 260 kb in human chromosome 19q13.3-13.4 (14 640 kb→274 990 kb). They are widely expressed in several tissues and mediate a wide range of critical physiological and pathological processes. Despite the recent developments in KLK research, elucidation of their physiological substrate repertoires remains a largely unfulfilled goal. Phage display, positional scanning and combinatorial peptide library screens have provided some valuable insights into the preferred specificities of these powerful enzymes. More recently, advances in proteomic technologies have enabled more systemic approaches towards identification of KLK substrates in a physiological setting. The advent of degradomic technologies has brought to light several putative physiological substrates and has allowed a deeper appreciation of the in vivo functional roles of KLKs. The aim of this review is to provide an overview of the different techniques that have been utilized towards the elucidation of the substrate specificities of these enzymes and elaborate on their emerging in vivo substrates.