scholarly journals Protease-activated receptor-2 signaling through β-arrestin-2 mediates Alternaria alkaline serine protease-induced airway inflammation

2018 ◽  
Vol 315 (6) ◽  
pp. L1042-L1057 ◽  
Author(s):  
Michael C. Yee ◽  
Heddie L. Nichols ◽  
Danny Polley ◽  
Mahmoud Saifeddine ◽  
Kasturi Pal ◽  
...  
Keyword(s):  
Airway Inflammation ◽  
Serine Protease ◽  
Protease Activity ◽  
Wild Type ◽  
Alkaline Serine Protease ◽  
Asthma Symptoms ◽  
Signaling Axis ◽  
Protease Activated Receptor 2

Alternaria alternata is a fungal allergen associated with severe asthma and asthma exacerbations. Similarly to other asthma-associated allergens, Alternaria secretes a serine-like trypsin protease(s) that is thought to act through the G protein-coupled receptor protease-activated receptor-2 (PAR2) to induce asthma symptoms. However, specific mechanisms underlying Alternaria-induced PAR2 activation and signaling remain ill-defined. We sought to determine whether Alternaria-induced PAR2 signaling contributed to asthma symptoms via a PAR2/β-arrestin signaling axis, identify the protease activity responsible for PAR2 signaling, and determine whether protease activity was sufficient for Alternaria-induced asthma symptoms in animal models. We initially used in vitro models to demonstrate Alternaria-induced PAR2/β-arrestin-2 signaling. Alternaria filtrates were then used to sensitize and challenge wild-type, PAR2−/− and β-arrestin-2−/− mice in vivo. Intranasal administration of Alternaria filtrate resulted in a protease-dependent increase of airway inflammation and mucin production in wild-type but not PAR2−/− or β-arrestin-2−/− mice. Protease was isolated from Alternaria preparations, and select in vitro and in vivo experiments were repeated to evaluate sufficiency of the isolated Alternaria protease to induce asthma phenotype. Administration of a single isolated serine protease from Alternaria, Alternaria alkaline serine protease (AASP), was sufficient to fully activate PAR2 signaling and induce β-arrestin-2−/−-dependent eosinophil and lymphocyte recruitment in vivo. In conclusion, Alternaria filtrates induce airway inflammation and mucus hyperplasia largely via AASP using the PAR2/β-arrestin signaling axis. Thus, β-arrestin-biased PAR2 antagonists represent novel therapeutic targets for treating aeroallergen-induced asthma.

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.

scholarly journals Reversible autoinhibitory regulation ofEscherichia colimetallopeptidase BepA for selective β-barrel protein degradation

2020 ◽  
Vol 117 (45) ◽  
pp. 27989-27996
Author(s):  
Yasushi Daimon ◽  
Shin-ichiro Narita ◽  
Ryoji Miyazaki ◽  
Yohei Hizukuri ◽  
Hiroyuki Mori ◽  
...  
Keyword(s):  
Active Site ◽  
Protease Activity ◽  
Underlying Mechanism ◽  
Zinc Ion ◽  
Wild Type ◽  
Protease Domain ◽  
Loop Region ◽  
Assembly Pathway

Escherichia coliperiplasmic zinc-metallopeptidase BepA normally functions by promoting maturation of LptD, a β-barrel outer-membrane protein involved in biogenesis of lipopolysaccharides, but degrades it when its membrane assembly is hampered. These processes should be properly regulated to ensure normal biogenesis of LptD. The underlying mechanism of regulation, however, remains to be elucidated. A recently solved BepA structure has revealed unique features: In particular, the active site is buried in the protease domain and conceivably inaccessible for substrate degradation. Additionally, the His-246 residue in the loop region containing helix α9 (α9/H246 loop), which has potential flexibility and covers the active site, coordinates the zinc ion as the fourth ligand to exclude a catalytic water molecule, thereby suggesting that the crystal structure of BepA represents a latent form. To examine the roles of the α9/H246 loop in the regulation of BepA activity, we constructed BepA mutants with a His-246 mutation or a deletion of the α9/H246 loop and analyzed their activities in vivo and in vitro. These mutants exhibited an elevated protease activity and, unlike the wild-type BepA, degraded LptD that is in the normal assembly pathway. In contrast, tethering of the α9/H246 loop repressed the LptD degradation, which suggests that the flexibility of this loop is important to the exhibition of protease activity. Based on these results, we propose that the α9/H246 loop undergoes a reversible structural change that enables His-246–mediated switching (histidine switch) of its protease activity, which is important for regulated degradation of stalled/misassembled LptD.

scholarly journals Protease Activity, Secretion, Cell Entry, Cytotoxicity, and Cellular Targets of Secreted Autotransporter Toxin of Uropathogenic Escherichia coli

2006 ◽  
Vol 74 (11) ◽  
pp. 6124-6134 ◽  
Author(s):  
Nathalie M. Maroncle ◽  
Kelsey E. Sivick ◽  
Rebecca Brady ◽  
Faye-Ellen Stokes ◽  
Harry L. T. Mobley
Keyword(s):  
Escherichia Coli ◽  
Serine Protease ◽  
Active Site ◽  
Protease Activity ◽  
Host Cells ◽  
Single Mutation ◽  
Wild Type ◽  
Passenger Domain ◽  
Hek 293

ABSTRACT The secreted autotransporter toxin (Sat), found predominantly in uropathogenic Escherichia coli, is a member of the SPATE (serine protease autotransporters of Enterobacteriaceae) family and, as such, has serine protease activity and causes cytopathic effects on various cell types. To assess the contribution of the serine protease active site to the mechanism of action of Sat, mutations were made in the first (S256I), in the second (S258A), or in both (S256I/S258A) serine residues within the active site motif. Mutations in the first or both serines reduced protease activity to background levels (P < 0.001); a single mutation in the second serine reduced activity by 60% compared to wild type (P < 0.001). After reversion of the S256I mutation to wild type (I256S), we confirmed S256 as the catalytically active serine. None of these mutations affected secretion of the mature passenger domain or release into the supernatant. The S256I mutation, however, abrogated the cytotoxicity of Sat on human bladder (UM-UC-3) and kidney (HEK 293) epithelial cells, characterized by rounding and elongation, respectively, and a high level of cell detachment. Moreover, S256 is essential for Sat to mediate cytoskeletal contraction and actin loss in host cells as well as to degrade specific membrane/cytoskeletal (fodrin and leukocyte function-associated molecule 1) and nuclear [microtubule-associated proteins, LIM domain-only protein 7, Rap GTPase-activating protein, poly(ADP-ribose) polymerase] proteins in vitro. Lastly, Sat was internalized by host cells and localized to the cytoskeletal fraction where membrane/cytoskeletal target proteins reside.

scholarly journals The Serine Protease Autotransporter Pic Modulates Citrobacter rodentium Pathogenesis and Its Innate Recognition by the Host

2015 ◽  
Vol 83 (7) ◽  
pp. 2636-2650 ◽  
Author(s):  
Kirandeep Bhullar ◽  
Maryam Zarepour ◽  
Hongbing Yu ◽  
Hong Yang ◽  
Matthew Croxen ◽  
...  
Keyword(s):  
Serine Protease ◽  
Shigella Flexneri ◽  
Citrobacter Rodentium ◽  
Wild Type ◽  
Content Type ◽  
The Family ◽  
Toll Like Receptor 2 ◽  
Severe Colitis

Bacterial pathogens produce a number of autotransporters that possess diverse functions. These include the family of serine protease autotransporters ofEnterobacteriaceae(SPATEs) produced by enteric pathogens such asShigella flexneriand enteroaggregativeEscherichia coli. Of these SPATEs, one termed “protein involved in colonization,” or Pic, has been shown to possess mucinase activityin vitro, but to date, its role inin vivoenteric pathogenesis is unknown. Testing apicnull (ΔpicC) mutant inCitrobacter rodentium, a natural mouse pathogen, found that theC. rodentiumΔpicCstrain was impaired in its ability to degrade mucinin vitrocompared to the wild type. Upon infection of mice, the ΔpicCmutant exhibited a hypervirulent phenotype with dramatically heavier pathogen burdens found in intestinal crypts. ΔpicCmutant-infected mice suffered greater barrier disruption and more severe colitis and weight loss, necessitating their euthanization between 10 and 14 days postinfection. Notably, the virulence of the ΔpicCmutant was normalized when thepicCgene was restored; however, a PicC point mutant causing loss of mucinase activity did not replicate the ΔpicCphenotype. Exploring other aspects of PicC function, the ΔpicCmutant was found to aggregate to higher levelsin vivothan wild-typeC. rodentium. Moreover, unlike the wild type, theC. rodentiumΔpicCmutant had a red, dry, and rough (RDAR) morphologyin vitroand showed increased activation of the innate receptor Toll-like receptor 2 (TLR2). Interestingly, theC. rodentiumΔpicCmutant caused a degree of pathology similar to that of wild-typeC. rodentiumwhen infecting TLR2-deficient mice, showing that despite its mucinase activity, PicC's major rolein vivomay be to limitC. rodentium's stimulation of the host's innate immune system.

scholarly journals Phenotypic Characterization of Resistant Val36 Variants of Hepatitis C Virus NS3-4A Serine Protease

2007 ◽  
Vol 52 (1) ◽  
pp. 110-120 ◽  
Author(s):  
Yi Zhou ◽  
Doug J. Bartels ◽  
Brian L. Hanzelka ◽  
Ute Müh ◽  
Yunyi Wei ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Serine Protease ◽  
Phenotypic Characterization ◽  
Replication Capacity ◽  
Wild Type ◽  
Protease Domain

ABSTRACT In patients chronically infected with hepatitis C virus (HCV) strains of genotype 1, rapid and dramatic antiviral activity has been observed with telaprevir (VX-950), a highly selective and potent inhibitor of the HCV NS3-4A serine protease. HCV variants with substitutions in the NS3 protease domain were observed in some patients during telaprevir dosing. In this study, purified protease domain proteins and reconstituted HCV subgenomic replicons were used for phenotypic characterization of many of these substitutions. V36A/M or T54A substitutions conferred less than eightfold resistance to telaprevir. Variants with double substitutions at Val36 plus Thr54 had ∼20-fold resistance to telaprevir, and variants with double substitutions at Val36 plus Arg155 or Ala156 had >40-fold resistance to telaprevir. An X-ray structure of the HCV strain H protease domain containing the V36M substitution in a cocomplex with an NS4A cofactor peptide was solved at a 2.4-Å resolution. Except for the side chain of Met36, the V36M variant structure is identical to that of the wild-type apoenzyme. The in vitro replication capacity of most variants was significantly lower than that of the wild-type replicon in cells, which is consistent with the impaired in vivo fitness estimated from telaprevir-dosed patients. Finally, the sensitivity of these replicon variants to alpha interferon or ribavirin remained unchanged compared to that of the wild-type.

scholarly journals The NS4A Protein of Hepatitis C Virus Promotes RNA-Coupled ATP Hydrolysis by the NS3 Helicase

2009 ◽  
Vol 83 (7) ◽  
pp. 3268-3275 ◽  
Author(s):  
Rudolf K. F. Beran ◽  
Brett D. Lindenbach ◽  
Anna Marie Pyle
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Serine Protease ◽  
Protease Activity ◽  
Rna Binding ◽  
Nonstructural Protein ◽  
Helicase Activity ◽  
Protease Domain

ABSTRACT Nonstructural protein 3 (NS3) is an essential replicative component of the hepatitis C virus (HCV) and a member of the DExH/D-box family of proteins. The C-terminal region of NS3 (NS3hel) exhibits RNA-stimulated NTPase and helicase activity, while the N-terminal serine protease domain of NS3 enhances RNA binding and unwinding by NS3hel. The nonstructural protein 4A (NS4A) binds to the NS3 protease domain and serves as an obligate cofactor for NS3 serine protease activity. Given its role in stimulating protease activity, we sought to determine whether NS4A also influences the activity of NS3hel. Here we show that NS4A enhances the ability of NS3hel to bind RNA in the presence of ATP, thereby acting as a cofactor for helicase activity. This effect is mediated by amino acids in the C-terminal acidic domain of NS4A. When these residues are mutated, one observes drastic reductions in ATP-coupled RNA binding and duplex unwinding by NS3. These same mutations are lethal in HCV replicons, thereby establishing in vitro and in vivo that NS4A plays an important role in the helicase mechanism of NS3 and its function in replication.

scholarly journals Interleukin-10 Is a Natural Suppressor of Cytokine Production and Inflammation in a Murine Model of Allergic Bronchopulmonary Aspergillosis

1997 ◽  
Vol 185 (6) ◽  
pp. 1089-1100 ◽  
Author(s):  
Gabriele Grünig ◽  
David B. Corry ◽  
Michael W. Leach ◽  
Brian W.P. Seymour ◽  
Viswanath P. Kurup ◽  
...  
Keyword(s):  
Airway Inflammation ◽  
Gene Knockout ◽  
Allergic Bronchopulmonary Aspergillosis ◽  
Interleukin 10 ◽  
Lung Cells ◽  
Wild Type ◽  
Outbred Mice ◽  
Ifn Γ

We have used interleukin-10 (IL-10) gene knockout mice (IL-10−/−) to examine the role of endogenous IL-10 in allergic lung responses to Aspergillus fumigatus Ag. In vitro restimulated lung cells from sensitized IL-10−/− mice produced exaggerated amounts of IL-4, IL-5, and interferon-γ (IFN-γ) compared with wild-type (WT) lung cells. In vivo, the significance of IL-10 in regulating responses to repeated A. fumigatus inhalation was strikingly revealed in IL-10−/− outbred mice that had a 50–60% mortality rate, while mortality was rare in similarly treated WT mice. Furthermore, IL-10−/− outbred mice exhibited exaggerated airway inflammation and heightened levels of IL-5 and IFN-γ in bronchoalveolar lavage (BAL) fluids. In contrast, the magnitude of the allergic lung response was similar in intranasally (i.n.) sensitized IL-10−/− and wild-type mice from a different strain (C57BL/6). Using a different route of priming (intraperitoneal) followed by one i.n. challenge we found that IL-10−/− C57BL/6 mice had heightened eosinophilic airway inflammation, BAL–IL-5 levels, and numbers of αβT cells in the lung tissues compared with WT mice. We conclude that IL-10 can suppress inflammatory Th2-like lung responses as well as Th1-like responses given the constraints of genetic background and route of priming.

Salmonella enterica Serovar Typhimurium HtrA: regulation of expression and role of the chaperone and protease activities during infection

Microbiology ◽  
2009 ◽  
Vol 155 (3) ◽  
pp. 873-881 ◽  
Author(s):  
Claire Lewis ◽  
Henrieta Skovierova ◽  
Gary Rowley ◽  
Bronislava Rezuchova ◽  
Dagmar Homerova ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Protease Activity ◽  
Pdz Domain ◽  
Proximal Promoter ◽  
Wild Type ◽  
Regulation Of Expression ◽  
Serovar Typhimurium

HtrA is a bifunctional stress protein required by many bacterial pathogens to successfully cause infection. Salmonella enterica serovar Typhimurium (S. Typhimurium) htrA mutants are defective in intramacrophage survival and are highly attenuated in mice. Transcription of htrA in Escherichia coli is governed by a single promoter that is dependent on σ E (RpoE). S. Typhimurium htrA also possesses a σ E-dependent promoter; however, we found that the absence of σ E had little effect on production of HtrA by S. Typhimurium. This suggests that additional promoters control expression of htrA in S. Typhimurium. We identified three S. Typhimurium htrA promoters. Only the most proximal promoter, htrAp3, was σ E dependent. The other promoters, htrAp1 and htrAp2, are probably recognized by the principal sigma factor σ 70. These two promoters were constitutively expressed but were also slightly induced by heat shock. Thus expression of htrA is different in S. Typhimurium and E. coli. The role of HtrA is to deal with misfolded/damaged proteins in the periplasm. It can do this either by degrading (protease activity) or folding/capturing (chaperone/sequestering, C/S, activity) the aberrant protein. We investigated which of these functions are important to S. Typhimurium in vitro and in vivo. Point or deletion mutants of htrA that encode variant HtrA molecules have been used in previous studies to investigate the role of different regions of HtrA in C/S and protease activity. These htrA variants were placed under the control of the S. Typhimurium htrAP123 promoters and expressed in a S. Typhimurium htrA mutant, GVB1343. Both wild-type HtrA and HtrA (HtrA S210A) lacking protease activity enabled GVB1343 to grow at high temperature (46 °C). Both molecules also significantly enhanced the growth/survival of GVB1343 in the liver and spleen of mice during infection. However, expression of wild-type HtrA enabled GVB1343 to grow to much higher levels than expression of HtrA S210A. Thus both the protease and C/S functions of HtrA operate in vivo during infection but the protease function is probably more important. Absence of either PDZ domain completely abolished the ability of HtrA to complement the growth defects of GVB1343 in vitro or in vivo.

Evidence that cell surface localization of serine protease activity facilitates cleavage of the protease activated receptor CDCP1

2018 ◽  
Vol 399 (9) ◽  
pp. 1091-1097
Author(s):  
Yaowu He ◽  
Janet C. Reid ◽  
Hui He ◽  
Brittney S. Harrington ◽  
Brittney Finlayson ◽  
...  
Keyword(s):  
Serine Protease ◽  
Protease Activity ◽  
Serine Proteases ◽  
Membrane Localization ◽  
Cub Domain ◽  
Serine Protease Activity ◽  
Surface Localization ◽  
Cell Surface Localization

Abstract The cellular receptor CUB domain containing protein 1 (CDCP1) is commonly elevated and functionally important in a range of cancers. CDCP1 is cleaved by serine proteases at adjacent sites, arginine 368 (R368) and lysine 369 (K369), which induces cell migration in vitro and metastasis in vivo. We demonstrate that membrane localization of serine protease activity increases efficacy of cleavage of CDCP1, and that both secreted and membrane anchored serine proteases can have distinct preferences for cleaving at CDCP1-R368 and CDCP1-K369. Approaches that disrupt membrane localization of CDCP1 cleaving serine proteases may interfere with the cancer promoting effects of CDCP1 proteolysis.

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