scholarly journals Taking the plunge: integrating structural, enzymatic and computational insights into a unified model for membrane-immersed rhomboid proteolysis

2010 ◽  
Vol 425 (3) ◽  
pp. 501-512 ◽  
Author(s):  
Sinisa Urban
Keyword(s):  
Serine Protease ◽  
Animal Cell ◽  
Membrane Surface ◽  
Cell Signalling ◽  
Integral Membrane Protein ◽  
Architectural Elements ◽  
Membrane Complex ◽  
Rhomboid Proteases

Rhomboid proteases are a fascinating class of enzymes that combine a serine protease active site within the core of an integral membrane protein. Despite having key roles in animal cell signalling and microbial pathogenesis, the membrane-immersed nature of these enzymes had long imposed obstacles to elucidating their biochemical mechanisms. But recent multidisciplinary approaches, including eight crystal structures, four computer simulations and nearly 100 engineered mutants interrogated in vivo and in vitro, are coalescing into an integrated model for one rhomboid orthologue in particular, bacterial GlpG. The protein creates a central hydrated microenvironment immersed below the membrane surface to support hydrolysis by its serine protease-like catalytic apparatus. Four conserved architectural elements in particular act as ‘keystones’ to stabilize this structure, and the lateral membrane-embedded L1 loop functions as a ‘flotation device’ to position the protease tilted in the membrane. Complex interplay between lateral substrate gating by rhomboid, substrate unwinding and local membrane thinning leads to intramembrane proteolysis of selected target proteins. Although far from complete, studies with GlpG currently offer the best prospect for achieving a thorough and sophisticated understanding of a simplified intramembrane protease.

scholarly journals Phenotypic Analysis of Mice Lacking the Tmprss2-Encoded Protease

2006 ◽  
Vol 26 (3) ◽  
pp. 965-975 ◽  
Author(s):  
Tom S. Kim ◽  
Cynthia Heinlein ◽  
Robert C. Hackman ◽  
Peter S. Nelson
Keyword(s):  
Serine Protease ◽  
Serine Proteases ◽  
Biological Activities ◽  
Type Ii ◽  
Phenotypic Analysis ◽  
Normal Prostate ◽  
Prostate Hyperplasia ◽  
Transmembrane Serine Protease

ABSTRACT Tmprss2 encodes an androgen-regulated type II transmembrane serine protease (TTSP) expressed highly in normal prostate epithelium and has been implicated in prostate carcinogenesis. Although in vitro studies suggest protease-activated receptor 2 may be a substrate for TMPRSS2, the in vivo biological activities of TMPRSS2 remain unknown. We generated Tmprss2 −/− mice by disrupting the serine protease domain through homologous recombination. Compared to wild-type littermates, Tmprss2 −/− mice developed normally, survived to adulthood with no differences in protein levels of prostatic secretions, and exhibited no discernible abnormalities in organ histology or function. Loss of TMPRSS2 serine protease activity did not influence fertility, reduce survival, result in prostate hyperplasia or carcinoma, or alter prostatic luminal epithelial cell regrowth following castration and androgen replacement. Lack of an observable phenotype in Tmprss2 −/− mice was not due to transcriptional compensation by closely related Tmprss2 homologs. We conclude that the lack of a discernible phenotype in Tmprss2 −/− mice suggests functional redundancy involving one or more of the type II transmembrane serine protease family members or other serine proteases. Alternatively, TMPRSS2 may contribute a specialized but nonvital function that is apparent only in the context of stress, disease, or other systemic perturbation.

scholarly journals The activity of superoxide-dismutase in animal cell culture CHO-K1 after treatment with fullerenol and mytomicine C

2009 ◽  
Vol 63 (3) ◽  
pp. 143-149 ◽  
Author(s):  
Visnja Bogdanovic ◽  
Marija Slavic ◽  
Jasminka Mrdjanovic ◽  
Slavica Solajic ◽  
Aleksandar Djordjevic
Keyword(s):  
Superoxide Dismutase ◽  
Mammalian Cells ◽  
Animal Cell ◽  
Water Soluble ◽  
Cytotoxic Agents ◽  
Antioxidant Defenses ◽  
Free Radical Scavengers ◽  
Sod Activity

Eukaryotic cell survives in predominantly reduced conditions. Homeostasis of cellular redox system is an imperative of cell surviving and its normal metabolism. ROS are well recognized for playing a dual role as both deleterious and beneficial species, since they can be either harmful or beneficial to living systems. These species are mutagenic compounds known to lead to DNA damage, favor cell transformation, and contribute to the development of a variety of malignant diseases. All the effects of oxidants are influenced by the cellular antioxidant defenses. This multilayer system consists of low molecular weight components and several antioxidant enzymes. Superoxide dismutases (SODs) are the only enzymes dismuting superoxide radicals. Mitomycin C, a cross-linking agent, demonstrated genotoxicity in all in vitro and in vivo test systems in mammalian cells and animals. Water-soluble fullerenes are well known as cytotoxic agents for many cell lines in vitro. At the other side, fullerenols are good free radical scavengers and antioxidants both in vitro and in vivo. This paper investigates the effects of fullerenol on survival and fullerenol/ /mytomicine (MMC) treatment on superoxide-dismutase (SOD) activity in CHO-K1 cells. Samples were treated 3 and 24 h with fullerenol (C60(OH)24) at concentration range 0.01-0.5 mg/mL and survival was monitored with dye exclusion test (DET). The activity of total SOD was estimated in samples treated with chosen concentrations of fullerenol and MMC (0.5 and 0.1 mg/mL) after 3 and 24 h of cell incubation. Increasing of C60(OH)24 concentration leads to decreasing of percent of surviving cells 3 and 24 h after incubation. The activity of total SOD enhanced with higher concentration of fullerenol, while decreased in the highest concentration at both experimental points. In samples treated with MMC, as well as in samples treated with fullerenol (0.0625 mg/mL) + MMC was noticed boost in total SOD activity in comparison with controls. Treatment with fullerenol decreased SOD activity in rest of samples treated with MMC. Decreased activity of superoxide-dismutase in almost all samples treated with fullerenol and MMC might be contributed to antioxidative properties of fullerenol. Increased enzyme level at concentration of 0.0625 mg/mL may be due to its prooxidative activity.

scholarly journals Investigating the ocular toxicity potential and therapeutic efficiency of in situ gel nanoemulsion formulations of brinzolamide

2020 ◽  
Vol 9 (4) ◽  
pp. 578-587
Author(s):  
Sima Talaei ◽  
Mohammad Mehdi Mahboobian ◽  
Mojdeh Mohammadi
Keyword(s):  
Intraocular Pressure ◽  
Membrane Surface ◽  
Chorioallantoic Membrane ◽  
In Situ Gel ◽  
Toxicity Potential ◽  
Vessel Injury ◽  
Hen’S Egg

Abstract Glaucoma is an ocular disease i.e. more common in older adults with elevated intraocular pressure and a serious threat to vision if it is not controlled. Due to the limitations regarding the conventional form of brinzolamide (Azopt®), two optimum formulations of in situ gel nanoemulsion were developed. To ensure the safety and efficacy of developed formulations for ocular drug delivery, the current study was designed. MTT assay was carried out on the human retinal pigmentation epithelial cells. To investigate the irritation potential of the chosen formulations, hen’s egg test-chorioallantoic membrane as a borderline test between in vivo and in vitro methods has been done. The modified Draize method was utilized to evaluate eye tolerance against the selected formulations. Intraocular pressure was measured by applying the prepared formulations to the eyes of normotensive albino rabbits in order to assess the therapeutic efficacy. Based on MTT test, cell viability for NE-2 at 0.1% and NE-1 at 0.1 and 0.5% concentrations was acceptable. The results of the hen’s egg test-chorioallantoic membrane test indicated no sign of vessel injury on the chorioallantoic membrane surface for both formulations. Also, during 24 h, both formulations were well-tolerated by rabbit eyes. The pharmacodynamics effects of formulations had no difference or were even higher than that of suspension in case of adding lower concentration (0.5%) of brinzolamide to the formulations. With regard to the results of the mentioned methods, our advanced formulations were effective, safe, and well-tolerated, thus can be introduced as an appropriate vehicle for ocular delivery of brinzolamide.

scholarly journals Stimulation in vitro of vitamin B12-dependent methionine synthase by polyamines

1996 ◽  
Vol 316 (2) ◽  
pp. 661-665 ◽  
Author(s):  
Susan H. KENYON ◽  
Anna NICOLAOU ◽  
Tamara AST ◽  
William A. GIBBONS
Keyword(s):  
Enzyme Activity ◽  
Vitamin B12 ◽  
Cell Signalling ◽  
Methionine Synthase ◽  
Polyamine Metabolism ◽  
Physiological Concentration ◽  
Methylation Pathway ◽  
Important Enzyme

Vitamin B12-dependent methionine synthase is an important enzyme for sulphur amino acid, folate polyamine metabolism, S-adenosylmethionine metabolism and also in the methylation pathway of DNA, RNA, proteins and lipids. Consequently, studies aiming at exploring the control and regulation of methionine synthase are of particular interest. Here we report the modulation of enzyme activity in vitro by polyamines. Although putrescine, cadaverine, spermine and spermidine all stimulated enzyme activity, the last two were the most potent, causing increases in enzyme activity up to 400%. The EC50 for spermine was determined as 8 μM and for spermidine 40 μM. The physiological concentration for spermine has been reported to be 15–19 μM. Spermine was found to increase both the Km and the Vmax with respect to methyltetrahydrofolate for the enzyme. These data support the hypothesis that spermine and spermidine are feedback regulators of methionine synthase both in vivo and in vitro and are consistent with the polyamines' regulating cell signalling pathways.

Molecular targets of nitric-oxide-donating aspirin in cancer

2005 ◽  
Vol 33 (4) ◽  
pp. 701-704 ◽  
Author(s):  
K. Kashfi ◽  
B. Rigas
Keyword(s):  
Nitric Oxide ◽  
Cell Signalling ◽  
Nuclear Factor Κb ◽  
Mitogen Activated Protein Kinase ◽  
Inhibition Of Proliferation ◽  
Mitogen Activated Protein ◽  
Nitric Oxide Synthase 2 ◽  
Oxide Synthase

Nitric-oxide-donating aspirin (NO-ASA), consisting of ASA (aspirin) plus an -ONO2 moiety linked to it via a molecular spacer, is a new drug for cancer prevention. NO-ASA seems to overcome the low potency and toxicity of traditional ASA. The -ONO2 moiety is responsible for releasing NO, and it appears to be required for biological activity. In studies in vitro, NO-ASA inhibits the growth of colon, pancreatic, prostate, lung, skin, leukaemia and breast cancer cells, and is up to 6000-fold more potent than traditional ASA. This effect is owing to cell kinetics [inhibition of proliferation, induction of apoptosis (multiple criteria) and blocking the G1 to S cell-cycle transition] and cell signalling [inhibition of Wnt signalling (IC50=0.2 μM), inhibition of NF-κB (nuclear factor κB) activation (IC50=7.5 μM), inhibition of nitric oxide synthase-2 expression (IC50=48 μM), inhibition of MAPK (mitogen-activated protein kinase) signalling (IC50=10 μM) and induction of cyclo-oxygenase-2 at approx. 10 μM]. In studies in vivo, NO-ASA inhibits intestinal carcinogenesis in Min mice (tumour multiplicity was reduced by 59% after 3 weeks, with no effect in control animals and no side effects) and in the N-nitrosobis(2-oxopropyl)amine model of pancreatic cancer, where there was an 89% reduction in NO-ASA (3000 p.p.m. in the diet)-treated animals (P<0.001). There was no statistically significant effect by traditional ASA at equimolar doses. Our data indicate that NO-ASA is a highly promising agent for the prevention and/or treatment of cancer.

scholarly journals Mesenchymal stem cells express serine protease inhibitor to evade the host immune response

Blood ◽  
2011 ◽  
Vol 117 (4) ◽  
pp. 1176-1183 ◽  
Author(s):  
Najib El Haddad ◽  
Dean Heathcote ◽  
Robert Moore ◽  
Sunmi Yang ◽  
Jamil Azzi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
T Cell ◽  
Protease Inhibitor ◽  
Serine Protease ◽  
Serine Protease Inhibitor ◽  
Cytotoxic T Cell ◽  
Wild Type

Abstract Clinical trials using mesenchymal stem cells (MSCs) have been initiated worldwide. An improved understanding of the mechanisms by which allogeneic MSCs evade host immune responses is paramount to regulating their survival after administration. This study has focused on the novel role of serine protease inhibitor (SPI) in the escape of MSCs from host immunosurveillance through the inhibition of granzyme B (GrB). Our data indicate bone marrow–derived murine MSCs express SPI6 constitutively. MSCs from mice deficient for SPI6 (SPI6−/−) exhibited a 4-fold higher death rate by primed allogeneic cytotoxic T cells than did wild-type MSCs. A GrB inhibitor rescued SPI6−/− MSCs from cytotoxic T-cell killing. Transduction of wild-type MSCs with MigR1-SPI6 also protected MSCs from cytotoxic T cell–mediated death in vitro. In addition, SPI6−/− MSCs displayed a shorter lifespan than wild-type MSCs when injected into an allogeneic host. We conclude that SPI6 protects MSCs from GrB-mediated killing and plays a pivotal role in their survival in vivo. Our data could serve as a basis for future SPI-based strategies to regulate the survival and function of MSCs after administration and to enhance the efficacy of MSC-based therapy for diseases.

PIVL, a snake venom Kunitz-type serine protease inhibitor, inhibits in vitro and in vivo angiogenesis

2014 ◽  
Vol 95 ◽  
pp. 149-156 ◽  
Author(s):  
Maram Morjen ◽  
Stéphane Honoré ◽  
Amine Bazaa ◽  
Zaineb Abdelkafi-Koubaa ◽  
Ameneallah Ellafi ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Serine Protease ◽  
Snake Venom ◽  
Serine Protease Inhibitor ◽  
Kunitz Type

A reinvestigation of the function of the mammalian urinary bladder

1977 ◽  
Vol 232 (3) ◽  
pp. F187-F195 ◽  
Author(s):  
S. A. Lewis
Keyword(s):  
Urinary Bladder ◽  
Membrane Surface ◽  
Apical Cell ◽  
Membrane Damage ◽  
In Vitro Experiments ◽  
Apical Cell Membrane ◽  
Cell Membrane Damage ◽  
Using Data

The function of adult mammalian urinary bladder is evaluated in light of recent in vitro experiments. The discrepancy between in vivo and in vitro experimental results is examined and a possible solution proposed. Techniques for eliminating edge damage and measuring apical membrane surface area are described. A new chamber design for microelectrode studies is illustrated. The possibility of apical cell membrane damage caused by microelectrodes is critically examined and tested using the polyene antibiotic Nystatin. Using data from transepithelial and microelectrode experiments, a model for net Na+ transport across the bladder is proposed and then critically analyzed. The possible clinical implications of the in vitro experiments are briefly discussed.

scholarly journals Novel and Modified Neutrophil Elastase Inhibitor Loaded in Topical Formulations for Psoriasis Management

Pharmaceutics ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 358 ◽  
Author(s):  
Andreia Nunes ◽  
Joana Marto ◽  
Lídia Maria Gonçalves ◽  
Sandra Simões ◽  
Rita Félix ◽  
...  
Keyword(s):  
Serine Protease ◽  
Neutrophil Elastase ◽  
In Vitro Cytotoxicity ◽  
Human Neutrophil Elastase ◽  
Therapeutic Effects ◽  
Neutrophil Elastase Inhibitor ◽  
Analytical Methodology ◽  
Elastase Inhibitor

Human neutrophil elastase (HNE) is a serine protease that degrades matrix proteins. An excess of HNE may trigger several pathological conditions, such as psoriasis. In this work, we aimed to synthesize, characterize and formulate new HNE inhibitors with a 4-oxo-β-lactam scaffold with less toxicity, as well as therapeutic index in a psoriasis context. HNE inhibitors with 4-oxo-β-lactam scaffolds were synthesized and characterized by NMR, FTIR, melting point, mass spectrometry and elemental analysis. In vitro cytotoxicity and serine protease assays were performed. The compound with the highest cell viability (AAN-16) was selected to be incorporated in an emulsion (AAN-16 E) and in a microemulsion (AAN-16 ME). Formulations were characterized in terms of organoleptic properties, pH, rheology, droplet size distribution, in vitro drug release and in vivo psoriatic activity. All compounds were successfully synthesized according to analytical methodology, with good yields. Both formulations presented suitable physicochemical properties. AAN-16 E presented the most promising therapeutic effects in a murine model of psoriasis. Overall, new HNE inhibitors were synthesized with high and selective activity and incorporated into topical emulsions with potential to treat psoriasis.

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