Kunitz-type serine protease inhibitors are a class of ubiquitous protease inhibitors, which play important roles in various life activities. The structures of such inhibitors are generally stable, and are usually characterized by the presence of one or several Kunitz domains in tandem, which are able to bind to serine proteases in a manner similar to substrate binding, thereby inhibiting enzyme activity. In terms of function, Kunitz-type serine protease inhibitors are involved in processes such as blood coagulation and fibrinolysis, tumor immunity, inflammation regulation, and resistance to bacterial and fungal infections. This article summarizes the advances of Kunitz-type serine protease inhibitors and provides new ideas for the development of novel Kunitz-type serine protease inhibitors.
Protease Inhibitors ; Serine Proteases ; Serine Proteinase Inhibitors

SKPI (shrimp Kunitz-type protease inhibitor) from Marsupenaeus japonicus is a member of serine protease inhibitors which play an important role in the arthropod immunity. To fully understand its function in the innate immunity of shrimp, the skpi gene was cloned into a modified pPIC9K vector with a 6-His tag and expressed by Pichia pastoris GS115. The secretory SKPI was purified from the medium with high purity by using Ni Sepharose High Performance. This results also indicated that the purified SKPI could inhibit the activity of trypsin specifically.
Animals ; Aprotinin ; biosynthesis ; genetics ; isolation & purification ; Pandalidae ; chemistry ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Serine Proteinase Inhibitors ; biosynthesis ; genetics ; Trypsin Inhibitors

Animals ; Atherosclerosis ; Humans ; Serine Proteinase Inhibitors ; Serpins

BACKGROUND: Serine protease promotes desquamatation of the stratum corneum and this is controlled by serine protease inhibitors (SPI). After disruption of the skin barrier, signals for barrier recovery are started with the activation of cytokines and a migration of calcium ions. On the other hand, the protease-activated receptor-2 (PAR-2) pathway is initiated as a negative signal. As the pH of the stratum corneum become neutral, activated serine protease and PAR-2 inhibit the secretion of lamellar bodies and the formation of the lamellar structure. OBJECTIVE: We wanted to screen noble synthetic peptides and identify the efficacy of a selected peptide, Palmitic acid-Lysine Threonine Threonine Lysine Serine (Pal-KTTKS), on PAR-2 in vitro and in vivo, and a clinical study was performed. METHODS: in vitro:Changes of the intracellular calcium ion concentration were measured in cultured HaCaT cells by fluorescence imaging according to treatment with sample peptides and trypsin. in vivo animal study:The efficacy of 2% Pal-KTTKS cream as a selected noble peptide was evaluated in an oxazolone-induced atopic dermatitis animal model. Clinical study:A total of twenty three atopic dermatitis patients applied 2.5% Pal-KTTKS peptide-containing cream on the one side of their extremities and pseudo-ceramide containing moisturizer on the other side of the extremities as a control twice a day for 4 weeks. Clinical improvement was evaluated by the Eczema Area Severity Index (EASI) score, a subject questionnaire and comparison of photographs. RESULTS: Suppression of the intracellular calcium concentration via PAR-2 inhibition was noted in the Pal-KTTKS peptide treated cultured HaCaT cells. In the oxazolone-induced atopic dermatitis hairless mice model, 2% Pal-KTTKS peptide containing lotion was more effective than vehicle lotion only. In the atopic dermatitis patients, the sites treated with 2.5% Pal-KTTKS peptide-containing cream showed better improvement for the EASI score, the subject questionnaire and the clinical photographs as compared to that of the control sites. There were no remarkable side effects related to the treatment. CONCLUSION: A PAR-2 inhibitor-containing topical agent would be an effective and safe modality for treating atopic dermatitis.
Animals ; Calcium ; Cytokines ; Dermatitis, Atopic ; Eczema ; Extremities ; Hand ; Humans ; Hydrogen-Ion Concentration ; Ions ; Lysine ; Mice ; Mice, Hairless ; Models, Animal ; Oligopeptides ; Optical Imaging ; Peptides ; Serine ; Serine Proteases ; Serine Proteinase Inhibitors ; Skin ; Threonine ; Trypsin ; Surveys and Questionnaires

BACKGROUND: Urinary trypsin inhibitor (UTI), which is speculated to have anti-inflammatory effects, is one of serine protease inhibitors found in human urine and blood. The present study was conducted to clarify the effect of urinary trypsin inhibitor (UTI) on human neutrophil activation and its intracellular signaling mechanism in vitro. METHODS: To assess the possible interactions between UTI and lipopolysaccharide (LPS) in neutrophil activation, neutrophils from human blood were incubated with varying concentrations of UTI (1, 10, 100, 1,000 and 10,000 U/ml) plus LPS (100 ng/ml) or LPS alone in 24-well plates (5 x 106 cells/well). We measured protein levels for interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-alpha) using enzyme-linked immunosorbent assay (ELISA) kits after 4 hours of incubation period. To elucidate the intracellular signaling pathway, we also measured the levels of phosphorylation of p38, ERK1/2 and JNK via Western blot analysis. Moreover, the nuclear levels of nuclear factor-kappa B (NF-kappaB) were determined with electrophoretic mobility shift assays (EMSA). RESULTS: UTI decreased the expression of inflammatory cytokines, including TNF-alpha and IL-6, and activation of intracellular signaling pathways, such as JNK, but not P38, ERK1/2 and nuclear translocation of NF-kappaB. CONCLUSIONS: UTI can attenuate LPS-induced neutrophil responses and may partially contribute to the treatment of neutrophil-mediated inflammatory diseases.
Blotting, Western ; Cytokines ; Electrophoretic Mobility Shift Assay ; Enzyme-Linked Immunosorbent Assay ; Glycoproteins ; Humans ; Interleukin-6 ; Interleukins ; Mitogen-Activated Protein Kinases ; Neutrophil Activation ; Neutrophils ; Phosphorylation ; Serine Proteinase Inhibitors ; Trypsin ; Tumor Necrosis Factor-alpha

Proteinase inhibitors are widely distributed in many living organisms and play crucial roles in many biological processes, particularly in regulating the proteinase activity spatially and temporally. However, The Kazal family of serine protease inhibitors is one of the most important and extensively studied protease inhibitor families. This type of protease inhibitor normally consists of one or several domains. Every domain has a highly conserved sequence structure and molecular conformation. It is found that contact residues are hyper variable, which are responsible for the interaction of inhibitors and proteinases. Most of them are in the solvent exposed loop. But P1 residue is the key active site of the interaction between inhibitor and enzyme. The types of the amino acid at P1 site likely play an important role in causing different inhibitory activity. The substitutions at the contact residues cause significant effects on the association constant. By using the Laskowski algorithm, the Ki values of a Kazal domain against six serine proteinases can be predicted from the domain' s sequence alone. At present there are many Kazal proteinase inhibitors found in the organisms, which show important biological functions. This article gives a comprehensive review of the newer developments in the characters and the interaction of the Kazal-type inhibitors.
Amino Acid Sequence ; Models, Molecular ; Molecular Sequence Data ; Protein Structure, Tertiary ; Serine Proteinase Inhibitors ; chemistry ; physiology

BACKGROUND AND OBJECTIVES: The nasal epithelium is the first barrier encountered by airborne allergens and an active participant in airway inflammation. Fungi have been increasingly recognized as important pathogens in sinusitis and consists of several allergenic proteins. We hypothesized that fungi induce the release of inflammatory mediators, and tried to find out the mechanism of epithelial cell activation. SUBJECTS AND METHOD: The epithelial cells of nasal polyp were obtained from patients and stimulated with Alternaria, Aspergillus, and Cladosporium. Interleukin-8 (IL-8), granulocyte-macrophage colony stimulating factor (GM-CSF), regulated on activation and normal T expressed and secreted (RANTES) were measured to determine the activation of epithelial cells. Nasal epithelial cell activation was inhibited with serine and cystein protease inhibitors. Reverse transcriptase-polymerase chain reaction (RT-PCR) test for protease-activated receptors (PARs) mRNA expression in nasal epithelial cells were performed. RESULTS: Fungi enhanced the production of chemical mediators from nasal epithelial cells. Serine protease inhibitors inhibited the activation of nasal epithelial cells. When nasal epithelial cells were activated, PAR2 and PAR3 mRNAs were more strongly expressed than non-activated cells. CONCLUSION: Serine proteases in fungi interact with nasal epithelial cells and enhance the production of inflammatory cytokines. PARs might play a role in the process of epithelial cell activation.
Allergens ; Alternaria ; Aspergillus ; Cladosporium ; Colony-Stimulating Factors ; Cytokines ; Epithelial Cells* ; Fungi* ; Humans ; Inflammation ; Interleukin-8 ; Nasal Mucosa ; Nasal Polyps ; Protease Inhibitors ; Receptors, Proteinase-Activated ; RNA, Messenger ; Serine ; Serine Proteases ; Serine Proteinase Inhibitors ; Sinusitis

Hespintor is an unknown function protein that was got from hepatoblastoma cell lines HepG2 by suppression subtractive hybridization technique (SSH), sequence analysis showed that the protein is a new member of secretory type of Kazal type serine protease inhibitor (Serpin) family, and has high homology with esophageal cancer related gene 2 (ECRG2). The coding sequence of Hespintor's Kazal domain was subcloned into prokaryotic expression vector pET-40b(+), then transformed into Rosetta (DE3). A recombinant protein about 42 kDa in the form of inclusion body was optimization expressed by inducing with 0.25 mmol/L IPTG, 30 degrees C for 5 h. and its specificity was confirmed via Western blotting. The recombinant protein was purified by metal chelate affinity chromatography (MCAC) and anion-exchange chromatography. The preliminary experimental result showed that the recombinant protein can inhibit trysin hydrolysis activity specifically. The result clearly demonstrated that Hespintor, as a novel member of Serpin, would be valuable in developing anti-tumor agents.
Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Hep G2 Cells ; Humans ; Proteinase Inhibitory Proteins, Secretory ; Recombinant Proteins ; biosynthesis ; genetics ; Serine Peptidase Inhibitors, Kazal Type ; Serine Proteinase Inhibitors ; biosynthesis ; classification ; genetics

BACKGROUND AND OBJECTIVES: Allergic fungal sinusitis is characterized by allergic mucin which includes eosinophils and fungal hyphae. But we don't know why eosinophils are accumulated at the nasal secretion and activated, so authors tried to explain the pathophysiologic function of eosinophils by directly stimulating eosinophils with fungal antigens. SUBJECTS AND METHOD: Eosinophils were isolated from healthy volunteers and stimulated with extracts from 5 common fungal species (Alternaria, Aspergillus, Candida, Cladosporium, and Penicillium). Superoxide production and eosinophil derived neurotoxin (EDN) were measured to determine whether fungi activated eosinophils. An inhibition study was done using serine and cystein protease inhibitors. RESULTS: When cultured with fungal antigens, eosinophils produced superoxide by Alternaria and Cladosporium but only Alternaria induced EDN production. Serine protease inhibitors (PMSF, Pefabloc?) and heat treatment of fungi significantly inhibited the activation of eosinophils but the cystein protease inhibitor (E-64) wasn't inhibited. CONCLUSION: Eosinophils are directly activated by Alternaria and their activity was inhibited by serine protease inhibitors. In AFS, fungal serine proteases may activate eosinophils which play important roles in the pathogenesis of AFS, resulting in the destruction of fungi and respiratory epithelial cells.
Alternaria ; Antigens, Fungal ; Aspergillus ; Candida ; Cladosporium ; Eosinophil-Derived Neurotoxin ; Eosinophils* ; Epithelial Cells ; Fungi* ; Healthy Volunteers ; Hot Temperature ; Hyphae ; Mucins ; Protease Inhibitors ; Serine ; Serine Proteases ; Serine Proteinase Inhibitors ; Sinusitis ; Superoxides

New antibiotics with novel modes of action and structures are urgently needed to combat the emergence of multidrug-resistant bacteria. Bacterial signal peptidase I (SPase I) is an indispensable enzyme responsible for cleaving the signal peptide of preprotein to release the matured proteins. Increasing evidence suggests that SPase I plays a crucial role in bacterial pathogenesis by regulating the excretion of a variety of virulent factors, maturation of quorum sensing factor and the intrinsic resistance against beta-lactams. Recently, breakthrough has been achieved in the understanding of three-dimensional structure of SPase I as well as the mechanism of enzyme-inhibitors interaction. Three families of inhibitors are identified, i.e. signal peptide derivatives, beta-lactams and arylomycins. In this article, we summarize the recent advance in the study of structure, activity and structure-activity relationship of SPase I inhibitors.
Animals ; Anti-Bacterial Agents ; pharmacology ; Bacteria ; drug effects ; Escherichia coli ; drug effects ; Membrane Proteins ; antagonists & inhibitors ; metabolism ; Oligopeptides ; chemistry ; pharmacology ; Serine Endopeptidases ; metabolism ; Serine Proteinase Inhibitors ; chemistry ; pharmacology ; Structure-Activity Relationship ; beta-Lactams ; antagonists & inhibitors