Atopic dermatitis (AD) is a common chronic inflammatory skin disease that results in significant morbidity. A hallmark of AD is disruption of the critical barrier function of upper epidermal layers, causatively linked to environmental stimuli, genetics, and infection, and a critical current target for the development of new therapeutic and prophylactic interventions. Staphylococcus aureus is an AD-associated pathogen producing virulence factors that induce skin barrier disruption in vivo and contribute to AD pathogenesis. We show, using immortalized and primary keratinocytes, that S. aureus protease SspA/V8 is the dominant secreted factor (in laboratory and AD clinical strains of S. aureus) inducing barrier integrity impairment and tight junction damage. V8-induced integrity damage was inhibited by an IL-1β-mediated mechanism, independent of effects on claudin-1. Induction of keratinocyte expression of the antimicrobial/host defense peptide human β-defensin 2 (hBD2) was found to be the mechanism underpinning this protective effect. Endogenous hBD2 expression was required and sufficient for protection against V8 protease-mediated integrity damage, and exogenous application of hBD2 was protective. This modulatory property of hBD2, unrelated to antibacterial effects, gives new significance to the defective induction of hBD2 in the barrier-defective skin lesions of AD and indicates therapeutic potential.
Effective treatment of reactive arthritis would ideally achieve both control of inflammation and eradication of persisting arthritogenic pathogens. We use a model of experimental Chlamydia trachomatis-induced arthritis (CtIA) to evaluate the effectiveness of nafamostat mesilate (NM), a serine protease inhibitor with complement-modifying effects and anticoagulant properties. To date clinical use of NM has largely been in Asia and has been primarily confined to inflammatory states such as pancreatitis.
Proteolytic activity of cell surface-associated MT1-MMP (MMP-14) is directly related to cell migration, invasion and metastasis. MT1-MMP is regulated as a proteinase by activation and conversion of the latent proenzyme into the active enzyme, and also via inhibition by tissue inhibitors of MMPs (TIMPs) and self-proteolysis. MT1-MMP is also regulated as a membrane protein through its internalization and recycling. Routine immunohistochemistry, flow cytometry, reverse transcription-PCR and immunoblotting methodologies do not allow quantitative imaging and assessment of the cell-surface levels of the active, TIMP-free MT1-MMP enzyme. Here, we developed a fluorescent reporter prototype that targets the cellular active MT1-MMP enzyme alone. The reporter (MP-3653) represents a liposome tagged with a fluorochrome and functionalized with a PEG chain spacer linked to an inhibitory hydroxamate warhead. Our studies using the MP-3653 reporter and its inactive derivative demonstrated that MP-3653 can be efficiently used not only to visualize the trafficking of MT1-MMP through the cell compartment, but also to quantify the femtomolar range amounts of the cell surface-associated active MT1-MMP enzyme in multiple cancer cell types, including breast carcinoma, fibrosarcoma and melanoma. Thus, the levels of the naturally expressed, fully functional, active cellular MT1-MMP enzyme are roughly equal to 1x105 molecules/cell, while these levels are in a 1x106 range in the cells with the enforced MT1-MMP expression. We suggest that the reporter we developed will contribute to the laboratory studies of MT1-MMP and then, ultimately, to the design of novel, more efficient prognostic approaches and personalized cancer therapies.
Despite a strict requirement for sterol removal for sperm to undergo acrosome exocytosis (AE), the mechanisms by which changes in membrane sterols are transduced into changes in sperm fertilization competence are poorly understood. We have previously shown in live murine sperm that the plasma membrane overlying the acrosome (APM) contains several types of micro-domains known as membrane rafts. When characterizing the membrane raft-associated proteomes, we identified phospholipase B (PLB), a calcium-independent enzyme exhibiting multiple activities. Here, we show that sperm surface PLB is activated in response to sterol removal. Both biochemical activity assays and immunoblots of sub-cellular fractions of sperm incubated with the sterol acceptor 2-hydroxypropyl-β-cyclodextrin (2-OHCD) confirmed the release of an active PLB fragment. Specific protease inhibitors prevented PLB activation, revealing a mechanistic requirement for proteolytic cleavage. Competitive inhibitors of PLB reduced both the ability of sperm to undergo AE and to fertilize oocytes in vitro, suggesting an important role in fertilization. This was reinforced by our finding that incubation with either protein concentrate released from 2-OHCD-treated sperm, or with recombinant PLB peptide corresponding to the catalytic domain, were both able to induce AE in the absence of other stimuli. Together, these results lead us to propose a novel mechanism by which sterol removal promotes membrane fusogenicity and AE, helping confer fertilization competence. Importantly, this mechanism provides a basis for the newly emerging model of AE in which membrane fusions occur during capacitation/transit through the cumulus, prior to any physical contact between the sperm and the oocyte’s zona pellucida.
Several splice variants of IgE exist in human plasma, including a variant called IgE-tailpiece (IgE-tp) that differs from classical IgE by the replacement of two carboxy-terminal amino acids with eight novel residues that include an ultimate cysteine. To date, the role of the secreted IgE-tp isoform in human immunity is unknown. We show that levels of IgE-tp are raised in helminth-infected donors, and that both the classical form of IgE (IgE-c) and IgE-tp interact with polymers of the serine protease inhibitor alpha-1-antitrypsin (A1AT). The association of IgE-tp with A1AT polymers in plasma protects the antibody from serine protease-mediated degradation, without affecting the functional interaction of IgE-tp with important receptors, including FcεR1. That polymers of A1AT protect IgE from degradation by helminth proteases may explain why these common and normally non-disease causing polymorphic variants of A1AT have been retained by natural selection. The observation that IgE can be complexed with polymeric forms of A1AT may therefore have important consequences for our understanding of the pathophysiology of pulmonary diseases that arise either as a consequence of A1AT-deficiency or through IgE-mediated type 1 hypersensitivity responses.
Reversal of mitochondrial defects with CSB-dependent serine protease inhibitors in patient cells of the progeroid Cockayne syndrome
- Proceedings of the National Academy of Sciences of the United States of America
- Published over 2 years ago
UV-sensitive syndrome (UV(S)S) and Cockayne syndrome (CS) are human disorders caused by CSA or CSB gene mutations; both conditions cause defective transcription-coupled repair and photosensitivity. Patients with CS also display neurological and developmental abnormalities and dramatic premature aging, and their cells are hypersensitive to oxidative stress. We report CSA/CSB-dependent depletion of the mitochondrial DNA polymerase-γ catalytic subunit (POLG1), due to HTRA3 serine protease accumulation in CS, but not in UV(s)S or control fibroblasts. Inhibition of serine proteases restored physiological POLG1 levels in either CS fibroblasts and in CSB-silenced cells. Moreover, patient-derived CS cells displayed greater nitroso-redox imbalance than UV(S)S cells. Scavengers of reactive oxygen species and peroxynitrite normalized HTRA3 and POLG1 levels in CS cells, and notably, increased mitochondrial oxidative phosphorylation, which was altered in CS cells. These data reveal critical deregulation of proteases potentially linked to progeroid phenotypes in CS, and our results suggest rescue strategies as a therapeutic option.
Background Patients who are chronically infected with hepatitis C virus (HCV) and who do not have a sustained virologic response after treatment with regimens containing direct-acting antiviral agents (DAAs) have limited retreatment options. Methods We conducted two phase 3 trials involving patients who had been previously treated with a DAA-containing regimen. In POLARIS-1, patients with HCV genotype 1 infection who had previously received a regimen containing an NS5A inhibitor were randomly assigned in a 1:1 ratio to receive either the nucleotide polymerase inhibitor sofosbuvir, the NS5A inhibitor velpatasvir, and the protease inhibitor voxilaprevir (150 patients) or matching placebo (150 patients) once daily for 12 weeks. Patients who were infected with HCV of other genotypes (114 patients) were enrolled in the sofosbuvir-velpatasvir-voxilaprevir group. In POLARIS-4, patients with HCV genotype 1, 2, or 3 infection who had previously received a DAA regimen but not an NS5A inhibitor were randomly assigned in a 1:1 ratio to receive sofosbuvir-velpatasvir-voxilaprevir (163 patients) or sofosbuvir-velpatasvir (151 patients) for 12 weeks. An additional 19 patients with HCV genotype 4 infection were enrolled in the sofosbuvir-velpatasvir-voxilaprevir group. Results In the three active-treatment groups, 46% of the patients had compensated cirrhosis. In POLARIS-1, the rate of sustained virologic response was 96% with sofosbuvir-velpatasvir-voxilaprevir, as compared with 0% with placebo. In POLARIS-4, the rate of response was 98% with sofosbuvir-velpatasvir-voxilaprevir and 90% with sofosbuvir-velpatasvir. The most common adverse events were headache, fatigue, diarrhea, and nausea. In the active-treatment groups in both trials, the percentage of patients who discontinued treatment owing to adverse events was 1% or lower. Conclusions Sofosbuvir-velpatasvir-voxilaprevir taken for 12 weeks provided high rates of sustained virologic response among patients across HCV genotypes in whom treatment with a DAA regimen had previously failed. (Funded by Gilead Sciences; POLARIS-1 and POLARIS-4 ClinicalTrials.gov numbers, NCT02607735 and NCT02639247 .).
Shock, sepsis, and multiorgan failure are associated with inflammation, morbidity, and high mortality. The underlying pathophysiological mechanism is unknown, but evidence suggests that pancreatic enzymes in the intestinal lumen autodigest the intestine and generate systemic inflammation. Blocking these enzymes in the intestine reduces inflammation and multiorgan dysfunction. We investigated whether enzymatic blockade also reduces mortality after shock. Three rat shock models were used here: hemorrhagic shock, peritonitis shock induced by placement of cecal material into the peritoneum, and endotoxin shock. One hour after initiation of hemorrhagic, peritonitis, or endotoxin shock, animals were administered one of three different pancreatic enzyme inhibitors-6-amidino-2-naphtyl p-guanidinobenzoate dimethanesulfate, tranexamic acid, or aprotinin-into the lumen of the small intestine. In all forms of shock, blockade of digestive proteases with protease inhibitor attenuated entry of digestive enzymes into the wall of the intestine and subsequent autodigestion and morphological damage to the intestine, lung, and heart. Animals treated with protease inhibitors also survived in larger numbers than untreated controls over a period of 12 weeks. Surviving animals recovered completely and returned to normal weight within 14 days after shock. The results suggest that the active and concentrated digestive enzymes in the lumen of the intestine play a central role in shock and multiorgan failure, which can be treated with protease inhibitors that are currently available for use in the clinic.
SERPINA3 (Serpin peptidase inhibitor clade A member 3), also known as a1-antichymotrypsin, is a serine protease inhibitor involved in a wide range of biological processes. Recently, it has been shown to be up-regulated in human placental diseases in association with a hypomethylation of the 5' region of the gene. In the present study, we show that the promoter of SERPINA3 is transcriptionally activated by three transcription factors (TFs) (SP1, MZF1 and ZBTB7B), the level of induction being dependent on the rs1884082 single nucleotide polymorphism (SNP) located inside the promoter, the T allele being consistently induced to a higher level than the G, with or without added TFs. When the promoter was methylated, the response to ZBTB7B was allele specific (the G allele was strongly induced, while the T allele was strongly down-regulated). We propose an adaptive model to explain the interest of such a regulation for placental function and homeostasis. Overexpression of SERPINA3 in JEG-3 cells, a trophoblast cell model, decreased cell adhesion to the extracellular matrix and to neighboring cells, but protects them from apoptosis, suggesting a way by which this factor could be deleterious at high doses. In addition, we show in different human populations that the T allele appears to predispose to Intra Uterine Growth Restriction (IUGR), while a G allele at a second SNP located in the second exon (rs4634) increases the risk of preeclampsia. Our results provide mechanistic views inside the involvement of SERPINA3 in placental diseases, through its regulation by a combination of epigenetic, genetic and TF-mediated regulations.
Bacterial flagella are cell locomotion and occasional adhesion organelles composed primarily of the polymeric protein flagellin, but to date have not been associated with any enzymatic function. Here, we report the bioinformatics-driven discovery of a class of enzymatic flagellins that assemble to form proteolytically active flagella. Originating by a metallopeptidase insertion into the central flagellin hypervariable region, this flagellin family has expanded to at least 74 bacterial species. In the pathogen, Clostridium haemolyticum, metallopeptidase-containing flagellin (which we termed flagellinolysin) is the second most abundant protein in the flagella and is localized to the extracellular flagellar surface. Purified flagellar filaments and recombinant flagellin exhibit proteolytic activity, cleaving nearly 1000 different peptides. With ~ 20,000 flagellin copies per ~ 10-μm flagella this assembles the largest proteolytic complex known. Flagellum-mediated extracellular proteolysis expands our understanding of the functional plasticity of bacterial flagella, revealing this family as enzymatic biopolymers that mediate interactions with diverse peptide substrates.So far no enzymatic activity has been attributed to flagellin, the major component of bacterial flagella. Here the authors use bioinformatic analysis and identify a metallopeptidase insertion in flagellins from 74 bacterial species and show that recombinant flagellin and flagellar filaments have proteolytic activity.