Proteinase-Mediated Macrophage Signaling in Psoriatic Arthritis

ObjectiveMultiple proteinases are present in the synovial fluid (SF) of an arthritic joint. We aimed to identify inflammatory cell populations present in psoriatic arthritis (PsA) SF compared to osteoarthritis (OA) and rheumatoid arthritis (RA), identify their proteinase-activated receptor 2 (PAR2) signaling function and characterize potentially active SF serine proteinases that may be PAR2 activators.MethodsFlow cytometry was used to characterize SF cells from PsA, RA, OA patients; PsA SF cells were further characterized by single cell 3’-RNA-sequencing. Active serine proteinases were identified through cleavage of fluorogenic trypsin- and chymotrypsin-like substrates, activity-based probe analysis and proteomics. Fluo-4 AM was used to monitor intracellular calcium cell signaling. Cytokine expression was evaluated using a multiplex Luminex panel.ResultsPsA SF cells were dominated by monocytes/macrophages, which consisted of three populations representing classical, non-classical and intermediate cells. The classical monocytes/macrophages were reduced in PsA compared to OA/RA, whilst the intermediate population was increased. PAR2 was elevated in OA vs. PsA/RA SF monocytes/macrophages, particularly in the intermediate population. PAR2 expression and signaling in primary PsA monocytes/macrophages significantly impacted the production of monocyte chemoattractant protein-1 (MCP-1). Trypsin-like serine proteinase activity was elevated in PsA and RA SF compared to OA, while chymotrypsin-like activity was elevated in RA compared to PsA. Tryptase-6 was identified as an active serine proteinase in SF that could trigger calcium signaling partially via PAR2.ConclusionPAR2 and its activating proteinases, including tryptase-6, can be important mediators of inflammation in PsA. Components within this proteinase-receptor axis may represent novel therapeutic targets.

Epoxy-α-Lapachone HasIn VitroandIn VivoAnti-Leishmania (Leishmania) amazonensis Effects and Inhibits Serine Proteinase Activity in This Parasite

ABSTRACTLeishmania(Leishmania)amazonensisis a protozoan that causes infections with a broad spectrum of clinical manifestations. The currently available chemotherapeutic treatments present many problems, such as several adverse side effects and the development of resistant strains. Natural compounds have been investigated as potential antileishmanial agents, and the effects of epoxy-α-lapachone onL. (L.)amazonensiswere analyzed in the present study. This compound was able to cause measurable effects on promastigote and amastigote forms of the parasite, affecting plasma membrane organization and leading to death after 3 h of exposure. This compound also had an effect in experimentally infected BALB/c mice, causing reductions in paw lesions 6 weeks after treatment with 0.44 mM epoxy-α-lapachone (mean lesion area, 24.9 ± 2.0 mm2), compared to untreated animals (mean lesion area, 30.8 ± 2.6 mm2) or animals treated with Glucantime (mean lesion area, 28.3 ± 1.5 mm2). In addition, the effects of this compound on the serine proteinase activities of the parasite were evaluated. Serine proteinase-enriched fractions were extracted from both promastigotes and amastigotes and were shown to act on specific serine proteinase substrates and to be sensitive to classic serine proteinase inhibitors (phenylmethylsulfonyl fluoride, aprotinin, and antipain). These fractions were also affected by epoxy-α-lapachone. Furthermore,in silicosimulations indicated that epoxy-α-lapachone can bind to oligopeptidase B (OPB) ofL. (L.)amazonensis, a serine proteinase, in a manner similar to that of antipain, interacting with an S1 binding site. This evidence suggests that OPB may be a potential target for epoxy-α-lapachone and, as such, may be related to the compound's effects on the parasite.

Prostatic trypsin-like kallikrein-related peptidases (KLKs) and other prostate-expressed tryptic proteinases as regulators of signalling via proteinase-activated receptors (PARs)

The prostate is a site of high expression of serine proteinases including members of the kallikrein-related peptidase (KLK) family, as well as other secreted and membrane-anchored serine proteinases. It has been known for some time that members of this enzyme family elicit cellular responses by acting directly on cells. More recently, it has been recognised that for serine proteinases with specificity for cleavage after arginine and lysine residues (trypsin-like or tryptic enzymes) these cellular responses are often mediated by cleavage of members of the proteinase-activated receptor (PAR) family – a four member sub-family of G protein-coupled receptors. Here, we review the expression of PARs in prostate, the ability of prostatic trypsin-like KLKs and other prostate-expressed tryptic enzymes to cleave PARs, as well as the prostate cancer-associated consequences of PAR activation. In addition, we explore the dysregulation of trypsin-like serine proteinase activity through the loss of normal inhibitory mechanisms and potential interactions between these dysregulated enzymes leading to aberrant PAR activation, intracellular signalling and cancer-promoting cellular changes.

Assembly in vitro of vitelline envelope components induced by a cortical alveolus sialoglycoprotein of eggs of the fish Tribolodon hakonensis

Assembly in vitro of vitelline envelope (VE) components, which were precipitated by 50–70% saturated ammonium sulphate from VE extracts, was induced by the action of a sialoglycoprotein that is immunohistochemically localised in cortical alveoli of fish eggs and has serine proteinase activity. The VE components consisted of major bands of molecular mass about 150–120, 110–100, 70 and 27 kDa in addition to about 20 minor bands and contained a chorionic transglutaminase, visualised as two fluorescent bands by monodansylcadaverine staining. The VE component assembly in vitro was Ca2+-dependent, not induced if the sialoglycoprotein was pretreated with a serine proteinase inhibitor, and inhibited by the presence of p-chloromercuribenzoate, iodoacetamide or L-cysteine in the reaction medium system. Electron microscopy revealed that assembly in vitro of the VE components consisted of aggregates of network sheets, consisting of branching and anastomosing thin (approximately 27–52 nm) and thick (approximately 137–376 nm) filamentous substances. Separation by SDS-PAGE showed that a considerable number of VE components participated in the assembly in vitro in various amounts. These results suggest at least partial reproduction of the phenomena that occur in the process of fertilisation envelope (FE) formation, and provide a new approach to investigation of the process of FE assembly in vitro.

The cysteine proteinases of Schistosoma mansoni cercariae

Based on substrate preferences, cercariae of Schistosoma mansoni were seen to express both cathepsin L and cathepsin B cysteine proteinases, although the former activity was many -fold greater. Two cathepsin L activities identified in cercarial extracts by zymography co-migrated with activities in extracts of 3 h and 24 h schisotosomula and in extracts of adult worms. Since these enzymes have been implicated in haemoglob in digestion by adult worms, they may perform a similar function in schistosomula. Immunolocalization using scanning electron micrographs showed that cathepsin L and cathepsin B proteinases were present in the cercarial post-acetabular glands. In addition, cercarial serine proteinase activities considered to facilitate skin penetration efficiently cleaved the substrates Z-Gly-Pro-Arg-NHMec and Z-Gly-Pro-Lys-NHMec. Cercariae release most of this serine proteinase activity when induced to secrete the contents of their acetabular glands. In contrast, newly transformed 3 h and 24 h schistosomula did not express this activity.