NAIP5/NLRC4 (neuronal apoptosis inhibitory protein 5/nucleotide oligomerization domain-like receptor family, caspase activation recruitment domain domain-containing 4) inflammasome activation by cytosolic flagellin results in caspase-1-mediated processing and secretion of IL-1β/IL-18 and pyroptosis, an inflammatory cell death pathway. Here, we found that although NLRC4, ASC, and caspase-1 are required for IL-1β secretion in response to cytosolic flagellin, cell death, nevertheless, occurs in the absence of these molecules. Cytosolic flagellin-induced inflammasome-independent cell death is accompanied by IL-1α secretion and is temporally correlated with the restriction of Salmonella Typhimurium infection. Despite displaying some apoptotic features, this peculiar form of cell death do not require caspase activation but is regulated by a lysosomal pathway, in which cathepsin B and cathepsin D play redundant roles. Moreover, cathepsin B contributes to NAIP5/NLRC4 inflammasome-induced pyroptosis and IL-1α and IL-1β production in response to cytosolic flagellin. Together, our data describe a pathway induced by cytosolic flagellin that induces a peculiar form of cell death and regulates inflammasome-mediated effector mechanisms of macrophages.
A trypsin was purified from pyloric caeca of pirarucu (Arapaima gigas). The effect of metal ions and protease inhibitors on its activity and its physicochemical and kinetic properties, as well its N-terminal sequence, were determined. A single band (28.0 kDa) was observed by SDS-PAGE. Optimum pH and temperature were 9.0 and 65°C, respectively. The enzyme was stable after incubation for 30 min in a wide pH range (6.0-11.5) and at 55°C. The kinetic parameters K m , k cat and k cat /K m were 0.47 ± 0.042 mM, 1.33 s À1 and 2.82 s À1 mM À1 , respectively, using BApNA as substrate. This activity was shown to be very sensitive to some metal ions, such as Fe 2+ , Hg 2+ , Zn 2+ , Al 3+ , Pb 2+ , and was highly inhibited by trypsin inhibitors. The trypsin N-terminal sequence IVGGYECPRNSVPYQ was found. The features of this alkaline peptidase suggest that it may have potential for industrial applications (e.g. food and detergent industries).
Accumulation of protein aggregates is a histopathological hallmark of several neurodegenerative diseases, but in most cases the aggregation occurs without defined mutations or clinical histories, suggesting that certain endogenous metabolites can promote aggregation of specific proteins. One example that supports this hypothesis is dopamine and its metabolites. Dopamine metabolism generates several oxidative metabolites that induce aggregation of α-synuclein, and represents the main etiology of Parkinson's diseases. Because dopamine and its metabolites are unstable and can be highly reactive, we investigated whether these molecules can also affect other proteins that are prone to aggregate, such as cellular prion protein (PrPC). In this study, we showed that dopamine treatment of neuronal cells reduced the number of viable cells and increased the production of reactive oxygen species (ROS) as demonstrated in previous studies. Overall PrPC expression level was not altered by dopamine treatment, but its unglycosylated form was consistently reduced at 100 μM of dopamine. At the same concentration, the level of phosphorylated mTOR and 4EBP1 was also reduced. Moreover, dopamine treatment decreased the solubility of PrPC, and increased its accumulation in autophagosomal compartments with concomitant induction of LC3-II and p62/SQSTM1 levels. In vitro oxidation of dopamine promoted formation of high-order oligomers of recombinant prion protein. These results suggest that dopamine metabolites alter the conformation of PrPC, which in turn is sorted to degradation pathway, causing autophagosome overload and attenuation of protein synthesis. Accumulation of PrPC aggregates is an important feature of prion diseases. Thus, this study brings new insight into the dopamine metabolism as a source of endogenous metabolites capable of altering PrPC solubility and its subcellular localization.
Human cytomegalovirus is a ubiquitous infectious agent that affects mainly immunosuppressed, fetuses, and newborns. The virus has several polymorphic regions, in particular in the envelope glycoproteins. The UL55 gene encodes the glycoprotein B that has a variable region, containing a furin cleavage site and according to the variability different genotypes are characterized. Here we investigated variability and existence of selective pressure on the UL55 variable region containing the furin cleavage site in 213 clinical sequences from patients worldwide. We showed the occurrence of positive selective pressure on gB codons 461 and 462, near the furin cleavage site. Cleavage analysis of synthesized peptides demonstrated that most mutations confer better cleavage by furin, suggesting that evolution is acting in order to increase the efficiency cleavage and supporting the hypothesis that gB processing is important in the host. We also demonstrated that peptides containing sequences, that characterize genotypes gB2 and 3, are differentially cleaved by furin. Our data demonstrate for the first time that variability in the cleavage site is related to degree of gB processing by furin.
We characterized the peroxidase mechanism of recombinant rat brain cytoglobin (Cygb) challenged by hydrogen peroxide, tert-butylhydroperoxide and by cumene hydroperoxide. The peroxidase mechanism of Cygb is similar to that of myoglobin. Cygb challenged by hydrogen peroxide is converted to a Fe4+ oxoferryl π cation, which is converted to Fe4+ oxoferryl and tyrosyl radical detected by direct continuous wave-electron paramagnetic resonance and by 3,5-dibromo-4-nitrosobenzene sulfonate spin trapping. When organic peroxides are used as substrates at initial reaction times, and given an excess of peroxide present, the EPR signals of the corresponding peroxyl radicals precede those of the direct tyrosyl radical. This result is consistent with the use of peroxide as a reducing agent for the recycling of Cygb high-valence species. Furthermore, we found that the Cygb oxidation by peroxides leads to the formation of amyloid fibrils. This result suggests that Cygb possibly participates in the development of degenerative diseases; our findings also support the possible biological role of Cygb related to peroxidase activity.
Thimet oligopeptidase ( EC 3.4.24.15 , TOP ) is a cytosolic mammalian zinc protease that can process a diversity of bioactive peptides. TOP has been pointed out as one of the main postproteasomal enzymes that process peptide antigens in the MHC class I presentation route. In the present study, we describe a fine regulation of TOP activity by hydrogen peroxide (H 2 O 2 ). Cells from a human embryonic kidney cell line ( HEK 293) underwent an ischemia/reoxygenation‐like condition known to increase H 2 O 2 production. Immediately after reoxygenation, HEK 293 cells exhibited a 32% increase in TOP activity, but no TOP activity was observed 2 h after reoxygenation. In another model, recombinant rat TOP ( rTOP ) was challenged by H 2 O 2 produced by rat liver mitoplasts ( RLM t) alone, and in combination with antimycin A, succinate, and antimycin A plus succinate. In these conditions, rTOP activity increased 17, 30, 32 and 38%, respectively. Determination of H 2 O 2 concentration generated in reoxygenated cells and mitoplasts suggested a possible modulation of rTOP activity dependent on the concentration of H 2 O 2 . The measure of pure rTOP activity as a function of H 2 O 2 concentration corroborated this hypothesis. The data fitted to an asymmetrical bell‐shaped curve in which the optimal activating H 2 O 2 concentration was 1.2 nM , and the maximal inhibition (75% about the control) was 1 μ m . Contrary to the oxidation produced by aging associated with enzyme oligomerization and inhibition, H 2 O 2 oxidation produced sulfenic acid and maintained rTOP in the monomeric form. Consistent with the involvement of rTOP in a signaling redox cascade, the H 2 O 2 ‐oxidized rTOP reacted with dimeric thioredoxin‐1 ( TR x‐1) and remained covalently bound to one subunit of TRx‐1.
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