Factor VII activating protease (FSAP) inhibits the outcome of ischemic stroke in mouse models.

Factor VII activating protease (FSAP) is a circulating serine protease, and individuals with the Marburg I (MI) single nucleotide polymorphism (SNP), which results in an inactive enzyme, have an increased risk of stroke. The outcome of ischemic stroke is more marked in FSAP-deficient mice compared to their wild-type (WT) counterparts. Plasma FSAP levels are raised in patients as well as mice after stroke. In vitro, FSAP promotes fibrinolysis by cleavage of fibrinogen, activates protease-activated receptors and decreases the cellular cytotoxicity of histones. Since these are desirable properties in stroke treatment, we tested the effect of recombinant serine protease domain of FSAP (FSAP-SPD) on ischemic stroke in mice. A combination of tissue plasminogen activator (tPA) and FSAP-SPD enhanced clot lysis, improved microvascular perfusion and neurological outcome and reduced infarct volumes in a mouse model of thromboembolic stroke. In the tail bleeding model FSAP-SPD treatment provoked a faster clotting time indicating that it has a pro-coagulant effect that is described before. FSAP-SPD improved stroke outcome and diminished the negative effects of co-treatment with tPA in the transient middle cerebral artery occlusion model. The inactive MI-isoform of FSAP did not have any effects in either model. In mice with FSAP deficiency there were minor differences in the outcomes of stroke but the treatment with FSAP-SPD was equally effective. Thus, FSAP represents a promising novel therapeutic strategy in the treatment of ischemic stroke that requires further evaluation.

A Novel Cleavage Pattern of Complement C5 Induced by Chlamydia trachomatis Infection via the Chlamydial Protease CPAF

Urogenital Chlamydia trachomatis infection is one of the most common bacterial sexually transmitted diseases globally. Untreated C. trachomatis infections can ascend to the upper genital tract and establish a series of severe complications. Previous studies using C3−/− and C5−/− mice models demonstrated that C3-independent activation of C5 occurred during C. trachomatis infection. However, the mechanism of how chlamydial infection activates C5 in the absence of C3 has yet to be elucidated. To delineate interactions between C5 and chlamydial infection, cleavage products in a co-incubation system containing purified human C5 and C. trachomatis-HeLa229 cell lysates were analyzed, and a novel cleavage pattern of C5 activation induced by C. trachomatis infection was identified. C5 was cleaved efficiently at the previously unidentified site K970, but was cleaved poorly at site R751. C5b was modified to C5bCt, which later formed C5bCt-9, which had enhanced lytic ability compared with C5b-9. The chlamydial serine protease CPAF contributed to C3-independent C5 activation during C. trachomatis infection. Nafamostat mesylate, a serine protease inhibitor with a good safety profile, had a strong inhibitory effect on C5 activation induced by chlamydial infection. These discoveries reveal the mechanism of C3-independent C5 activation induced by chlamydial infection, and furthermore provide a potential therapeutic target and drug for preventing tubal fibrosis caused by chlamydial infection.

Prevalence of the polymorphic H-ficolin (FCN3) genes and mannosebinding lectin-associated serine protease-2 (MASP2) in indigenous populations from the Russian Arctic regions

Lectins, being the main proteins of the lectin pathway activating the complement system, are encoded by polymorphic genes, wherein point mutations cause the protein conformation and expression to change, which turns out to have an effect on the functionality and ability to respond to the pathogen. In the current study, largescale data on the population genotype distribution of the genes for H-ficolin FCN3 rs28357092 and mannose-binding lectin-associated serine protease MASP2 rs72550870 among the indigenous peoples of the Russian Arctic regions (Nenets, Dolgans and Nganasans, a mixed population and Russians: a total sample was about 1000 newborns) have been obtained for the first time. Genotyping was carried out using RT-PCR. The frequency of the homozygous variant del/del FCN3 rs28357092 associated with the total absence of the most powerful activator of the lectin complement pathway, N-ficolin, was revealed; 0 % in the Nenets, 0.8 % in the Dolgans and Nganasans, and 3.5 % among the Russians ( p < 0.01). Analysis of the prevalence of the MASP2 genotypes has shown the predominance of the homozygous variant AA in all studied populations, which agrees with the available world data. The heterozygous genotype AG rs72550870 associated with a reduced level of protease was found to occur rarely in the Nenets, Dolgans and Nganasans compared to newborns of Caucasoid origin from Krasnoyarsk: 0.5 % versus 3.3 %, respectively. Moreover, among 323 examined Nenets, one AG carrier was identified, whereas in Russians, 16 out of 242 examined newborns were found to be AG carriers ( p < 0.001). A homozygous variant (GG) in total absence of protease with impaired binding of both MBL and ficolins was not detected in any of the 980 examined newborns. An additional analysis of infectious morbidity in Arctic populations allows one to find phenotypic characteristics related to a high functional activity of the lectin pathway of complement activation as an most important factor for the first-line of anti-infectious defense, including such new viral diseases as COVID-19.

Novel Feather Degrading Keratinases from Bacillus cereus Group: Biochemical, Genetic and Bioinformatics Analysis

In this study, five keratinolytic bacteria were isolated from poultry farm waste of Eastern Province, Saudi Arabia. The highest keratinase activity was obtained at 40–45 °C, pH 8–9, feather concentration 0.5–1%, and using white chicken feather as keratin substrate for 72 h. Enhancement of keratinase activity through physical mutagen UV radiation and/or chemical mutagen ethyl methanesulfonate (EMS) resulted in five mutants with 1.51–3.73-fold increased activity over the wild type. When compared with the wild type, scanning electron microscopy validated the mutants’ effectiveness in feather degradation. Bacterial isolates are classified as members of the S8 family peptidase Bacillus cereus group based on sequence analysis of the 16S rRNA and keratinase genes. Interestingly, keratinase KerS gene shared 95.5–100% identity to keratinase, thermitase alkaline serine protease, and thermophilic serine protease of the B. cereus group. D137N substitution was observed in the keratinase KerS gene of the mutant strain S13 (KerS13uv+ems), and also seven substitution variations in KerS26 and KerS26uv of strain S26 and its mutant S26uv. Functional analysis revealed that the subtilisin-like serine protease domain containing the Asp/His/Ser catalytic triad of KerS gene was not affected by the predicted substitutions. Prediction of physicochemical properties of KerS gene showed instability index between 17.5–19.3 and aliphatic index between 74.7–75.7, which imply keratinase stability and significant thermostability. The docking studies revealed the impact of substitutions on the superimposed structure and an increase in binding of mutant D137N of KerS13uv+ems (affinity: −7.17; S score: −6.54 kcal/mol) and seven mutants of KerS26uv (affinity: −7.43; S score: −7.17 kcal/mol) compared to the wild predicted structure (affinity: −6.57; S score: −6.68 kcal/mol). Together, the keratinolytic activity, similarity to thermostable keratinases, and binding affinity suggest that keratinases KerS13uv+ems and KerS26uv could be used for feather processing in the industry.

Oxidized Substrates of APEH as a Tool to Study the Endoprotease Activity of the Enzyme

APEH is a ubiquitous and cytosolic serine protease belonging to the prolyl oligopeptidase (POP) family, playing a critical role in the processes of degradation of proteins through both exo- and endopeptidase events. Endopeptidase activity has been associated with protein oxidation; however, the actual mechanisms have yet to be elucidated. We show that a synthetic fragment of GDF11 spanning the region 48–64 acquires sensitivity to the endopeptidase activity of APEH only when the methionines are transformed into the corresponding sulphoxide derivatives. The data suggest that the presence of sulphoxide-modified methionines is an important prerequisite for the substrates to be processed by APEH and that the residue is crucial for switching the enzyme activity from exo- to endoprotease. The cleavage occurs on residues placed on the C-terminal side of Met(O), with an efficiency depending on the methionine adjacent residues, which thereby may play a crucial role in driving and modulating APEH endoprotease activity.