Plasmin-mediated fibrinolysis enables macrophage migration in a murine model of inflammation.

Efficient migration of macrophages to sites of inflammation requires cell surface-bound plasmin(ogen). Here, we investigated the mechanisms underlying the deficits of plasmin(ogen)-mediated macrophage migration in 2 models: murine thioglycollate-induced peritonitis and in vitro macrophage migration. As previously reported, macrophage migration into the peritoneal cavity of mice in response to thioglycollate was significantly impaired in the absence of plasminogen. Fibrin(ogen) deposition was noted in the peritoneal cavity in response to thioglycollate, with a significant increase in fibrin(ogen) in the plasminogen-deficient mice. Interestingly, macrophage migration was restored in plasminogen-deficient mice by simultaneous imposition of fibrinogen deficiency. Consistent with this in vivo finding, chemotactic migration of cultured macrophages through a fibrin matrix did not occur in the absence of plasminogen. The macrophage requirement for plasmin-mediated fibrinolysis, both in vivo and in vitro, was negated by deletion of the major myeloid integrin αMß2-binding motif on the γ chain of fibrin(ogen). The study identifies a critical role of fibrinolysis in macrophage migration, presumably through the alleviation of migratory constraints imposed by the interaction of leukocytes with fibrin(ogen) through the integrin αMß2 receptor.

Integration of Two In-depth Quantitative Proteomics Approaches Determines the Kallikrein-related Peptidase 7 (KLK7) Degradome in Ovarian Cancer Cell Secretome.

Kallikrein-related peptidase 7 (KLK7) is a serine peptidase that is over expressed in ovarian cancer. In vitro functional analyses have suggested KLK7 to play a cancer progressive role, although monitoring of KLK7 expression has suggested a contradictory protective role for KLK7 in ovarian cancer patients. In order to help delineate its mechanism of action and thereby the functional roles, information on its substrate repertoire is crucial. Therefore, in this study a quantitative proteomics approach-PROtein TOpography and Migration Analysis Platform (PROTOMAP)-coupled with SILAC was used for in-depth analysis of putative KLK7 substrates from a representative ovarian cancer cell line, SKOV-3, secreted proteins. The Terminal Amine Isotopic Labeling of Substrates (TAILS) approach was used to determine the exact cleavage sites and to validate qPROTOMAP-identified putative substrates. By employing these two technically divergent approaches, exact cleavage sites on 16 novel putative substrates and two established substrates, matrix metalloprotease (MMP) 2 and insulin growth factor binding protein 3 (IGFBP3), were identified in the SKOV-3 secretome. Eight of these substrates were also identified on TAILS analysis of another ovarian cancer cell (OVMZ-6) secretome, with a further seven OVMZ-6 substrates common to the SKOV-3 qPROTOMAP profile. Identified substrates were significantly associated with the common processes of cell adhesion, extracellular matrix remodeling and cell migration according to the gene ontology (GO) biological process analysis. Biochemical validation supports a role for KLK7 in directly activating pro-MMP10, hydrolysis of IGFBP6 and cleavage of thrombospondin 1 with generation of a potentially bioactive N-terminal fragment. Overall, this study constitutes the most comprehensive analysis of the putative KLK7 degradome in any cancer to date, thereby opening new avenues for KLK7 research.

Mass spectrometry based proteomics analyses in kallikrein-related peptidase research: implications for cancer research and therapy.

INTRODUCTION: Kallikrein-related peptidases (KLKs) are a family of serine peptidases that are deregulated in numerous pathological conditions, with a multitude of KLK-mediated functional roles implicated in the progression of cancer. Advances in multidimensional mass spectrometry (MS)-based proteomics have facilitated the quantitative measurement of deregulated KLK expression in cancer, identifying certain KLKs, as well as their substrates, as potential cancer biomarkers. Areas covered: In this review, we discuss how these approaches have been utilized for KLK biomarker discovery and unbiased substrate determination in complex protein pools that mimic the in vivo extracellular microenvironment. Expert commentary: Although a limited number of studies have been performed, the quantity of information generated has greatly improved our understanding of the functional roles of KLKs in cancer progression. In addition, these data suggest additional means through which deregulated KLK expression may be targeted in cancer treatment, highlighting the potential therapeutic value of these state-of-the-art MS-based studies.

Live Imaging and Quantification of Neutrophil Extracellular Trap Formation.

NeutrophilExtracellular Trap (NET) formation (NETosis) is a unique process that occurs in response to numerous stimuli. To investigate NETosis, we created a method that can be used easily without the need for complex programming abilities and commercial software packages. This article describes a fully automated assay to quantify NETosis using fluorescence live imaging on an automated widefield inverted microscope. Herein, we describe (1) sample preparation, (2) required equipment for automated acquisition, and finally (3) analysis of NETosis using the readily available image analysis software Fiji (ImageJ2). This protocol can be adapted to evaluate NETosis after different stimuli, and can be easily modified to allow high-throughput acquisition and analysis using a multi-well plate format. Published 2021. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Neutrophil isolation and plate setup Basic Protocol 2: Microscope and acquisition setup for automated high throughput imaging Basic Protocol 3: Analysis of NETosis and apoptosis data.

The molecular function of kallikrein-related peptidase 14 demonstrates a key modulatory role in advanced prostate cancer.

Kallikrein-related peptidase 14 (KLK14) is one of the several secreted KLK serine proteases involved in prostate cancer (PCa) pathogenesis. While relatively understudied, recent reports have identified KLK14 as overexpressed during PCa development. However, the modulation of KLK14 expression during PCa progression and the molecular and biological functions of this protease in the prostate tumor microenvironment remain unknown. To determine the modulation of KLK14 expression during PCa progression, we analyzed the expression levels of KLK14 in patient samples using publicly available databases and immunohistochemistry. In order to delineate the molecular mechanisms involving KLK14 in PCa progression, we integrated proteomic, transcriptomic, and in vitro assays with the goal to identify substrates, related-signaling pathways, and functional roles of this protease. We showed that KLK14 expression is elevated in advanced PCa, and particularly in metastasis. Additionally, KLK14 levels were found to be decreased in PCa tissues from patients responsive to neoadjuvant therapy compared to untreated patients. Furthermore, we also identified that KLK14 expression reoccurred in patients who developed castrate-resistant PCa. The combination of proteomic and transcriptomic analysis as well as functional assays revealed several new KLK14 substrates (agrin, desmoglein 2, vitronectin, laminins) and KLK14-regulated genes (Interleukin 32, midkine, SRY-Box 9), particularly an involvement of the mitogen-activated protein kinase 1 and interleukin 1 receptor pathways, and an involvement of KLK14 in the regulation of cellular migration, supporting its involvement in aggressive features of PCa progression. In conclusion, our work showed that KLK14 expression is associated with the development of aggressive PCa suggesting that targeting this protease could offer a novel route to limit the progression of prostate tumors. Additional work is necessary to determine the benefits and implications of targeting/cotargeting KLK14 in PCa as well as to determine the potential use of KLK14 expression as a predictor of PCa aggressiveness or response to treatment.

Mass spectrometry-based determination of Kallikrein-related peptidase 7 (KLK7) cleavage preferences and subsite dependency.

The cleavage preferences of Kallikrein-related peptidase 7 (KLK7) have previously been delineated using synthetic peptide libraries of fixed length, or single protein chains and have suggested that KLK7 exerts a chymotryptic-like cleavage preference. Due to the short length of the peptides utilised, only a limited number of subsites have however been assessed. To determine the subsite preferences of KLK7 in a global setting, we used a mass spectrometry (MS)-based in-depth proteomics approach that utilises human proteome-derived peptide libraries of varying length, termed Proteomic Identification of protease Cleavage Sites (PICS). Consistent with previous findings, KLK7 was found to exert chymotryptic-like cleavage preferences. KLK7 subsite preferences were also characterised in the P2-P2' region, demonstrating a preference for hydrophobic residues in the non-prime and hydrophilic residues in the prime subsites. Interestingly, single catalytic triad mutant KLK7 (mKLK7; S195A) also showed residual catalytic activity (kcat/KM = 7.93 × 102 s-1M-1). Catalytic inactivity of KLK7 was however achieved by additional mutation in this region (D102N). In addition to characterising the cleavage preferences of KLK7, our data thereby also suggests that the use of double catalytic triad mutants should be employed as more appropriate negative controls in future investigations of KLK7, especially when highly sensitive MS-based approaches are employed.