Detection of subtilisin 3 and 6 in skin biopsies of cattle with clinically manifested bovine ringworm.

Trichophyton (T.) verrucosum is a highly pathogenic dermatophyte causing zoonotic bovine ringworm that is transmissible to humans. The virulence factors subtilisin (Sub)3 and Sub6 are discussed to contribute to disease manifestation but no protein expression study is available for T. verrucosum. We used customized antibodies (against Trichophyton-species, Sub3 and Sub6) to examine skin biopsies of infected cattle via immunofluorescence stainings. Both virulence factors Sub3 and 6 were solely expressed by conidia and not only found in epidermal but also in dermal and hair structures. The anti-T-antibody reliably detected the fungus and proved more sensitive compared to histological stains. LAY SUMMARY: We examined the zoonotic dermatophyte Trichophyton (T.) verrucosum in bovine skin and studied two important virulence factors called subtilisin (Sub)3 and Sub6 that T. verrucosum produces and secretes using immunolabeling.

Isolation and characterization of a Solanum tuberosum subtilisin-like protein with caspase-3 activity (StSBTc-3).

Plant proteases with caspase-like enzymatic activity have been widely studied during the last decade. Previously, we have reported the presence and induction of caspase-3 like activity in the apoplast of potato leaves during Solanum tuberosum- Phytophthora infestans interaction. In this work we have purified and identified a potato extracellular protease with caspase-3 like enzymatic activity from potato leaves infected with P. infestans. Results obtained from the size exclusion chromatography show that the isolated protease is a monomeric enzyme with an estimated molecular weight of 70 kDa approximately. Purified protease was analyzed by MALDI-TOF MS, showing a 100% of sequence identity with the deduced amino acid sequence of a putative subtilisin-like protease from S. tuberosum (Solgenomics protein ID: PGSC0003DMP400018521). For this reason the isolated protease was named as StSBTc-3. This report constitutes the first evidence of isolation and identification of a plant subtilisin-like protease with caspase-3 like enzymatic activity. In order to elucidate the possible function of StSBTc-3 during plant pathogen interaction, we demonstrate that like animal caspase-3, StSBTc-3 is able to produce in vitro cytoplasm shrinkage in plant cells and to induce plant cell death. This result suggest that, StSBTc-3 could exert a caspase executer function during potato- P. infestans interaction, resulting in the restriction of the pathogen spread during plant-pathogen interaction.

Categorisation of protein respiratory allergens: the case of Subtilisin.

Characterisation of the relative sensitizing potency of protein and chemical allergens remains challenging, particularly for materials causing allergic sensitization of the respiratory tract. There nevertheless remains an appetite, for priority setting and risk management, to develop paradigms that distinguish between individual respiratory allergens according to perceptions of the hazards and risks posed to human health. One manifestation thereof is recent listing of certain respiratory allergens as Substances of Very High Concern (SVHC) under the provisions of REACH (Registration, Evaluation, Authorisation and restriction of Chemicals). Although priority setting is a laudable ambition, it is important the process is predicated on evidence-based criteria that are transparent, understood and owned. The danger is that in the absence of rigorous criteria unwanted precedents can be created, and confidence in the process is compromised. A default categorisation of sensitisers as SVHC requiring assessment under the authorisation process is not desirable. We therefore consider here the value and limitations of selective assignment of certain respiratory allergens as being SVHC. The difficulties of sustaining such designations in a sound and equitable way is discussed in the context of the challenges that exist with respect to assessment of potency, and information available regarding the effectiveness of exposure-based risk management.

A plant alternative to animal caspases: subtilisin-like proteases.

Activities displaying caspase cleavage specificity have been well documented in various plant programmed cell death (PCD) models. However, plant genome analyses have not revealed clear orthologues of caspase genes, indicating that enzyme(s) structurally unrelated yet possessing caspase specificity have functions in plant PCD. Here, we review recent data showing that some caspase-like activities are attributable to the plant subtilisin-like proteases, saspases and phytaspases. These proteases hydrolyze a range of tetrapeptide caspase substrates following the aspartate residue. Data obtained with saspases implicate them in the proteolytic degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) during biotic and abiotic PCD, whereas phytaspase overproducing and silenced transgenics provide evidence that phytaspase regulates PCD during both abiotic (oxidative and osmotic stresses) and biotic (virus infection) insults. Like caspases, phytaspases and saspases are synthesized as proenzymes, which are autocatalytically processed to generate a mature enzyme. However, unlike caspases, phytaspases and saspases appear to be constitutively processed and secreted from healthy plant cells into the intercellular space. Apoplastic localization presumably prevents enzyme-mediated protein fragmentation in the absence of PCD. In response to death-inducing stimuli, phytaspase has been shown to re-localize to the cell interior. Thus, plant PCD-related proteases display both common (D-specific protein fragmentation during PCD) and distinct (enzyme structure and activity regulation) features with animal PCD-related proteases.

Subtilisin-like proteases in nematodes.

Cleavage by subtilisin-like proteases (subtilases) is an essential step in post-translational processing of proteins found in organisms ranging from yeast to mammals. Our knowledge of the diversity of this protease family in nematodes is aided by the rapid increase in sequence information, especially from the Brugia malayi genome project. Genetic studies of the subtilases in Caenorhabitis elegans give valuable insight into the biological function of these proteases in other nematode species. In this review, we focus on the subtilases in filarial nematodes as well as other parasitic and free-living nematodes in comparison to what is known in C. elegans. Topics to be addressed include expansion and diversity of the subtilase gene family during evolution, enhanced complexity created by alternative RNA splicing, molecular and biochemical characterization of the different subtilases and the challenges of designing subtilase-specific inhibitors for parasitic nematodes.

Effect of selected Ser/Ala and Xaa/Pro mutations on the stability and catalytic properties of a cold adapted subtilisin-like serine proteinase.

A subtilisin-like serine proteinase from a psychrotrophic Vibrio species (VPR) shows distinct cold adapted traits regarding stability and catalytic properties, while sharing high sequence homology with enzymes adapted to higher temperatures. Based on comparisons of sequences and examination of 3D structural models of VPR and related enzymes of higher temperature origin, five sites were chosen to be subject to site directed mutagenesis. Three serine residues were substituted with alanine and two residues in loops were substituted with proline. The single mutations were combined to make double and triple mutants. The single Ser/Ala mutations had a moderately stabilizing effect and concomitantly decreased catalytic efficiency. Introducing a second Ser/Ala mutation did not have additive effect on stability; on the contrary a double Ser/Ala mutant had reduced stability with regard to both wild type and single mutants. The Xaa/Pro mutations stabilized the enzyme and did also tend to decrease the catalytic efficiency more than the Ser/Ala mutations.

Molecular markers of serine protease evolution.

The evolutionary history of serine proteases can be accounted for by highly conserved amino acids that form crucial structural and chemical elements of the catalytic apparatus. These residues display non- random dichotomies in either amino acid choice or serine codon usage and serve as discrete markers for tracking changes in the active site environment and supporting structures. These markers categorize serine proteases of the chymotrypsin-like, subtilisin-like and alpha/beta-hydrolase fold clans according to phylogenetic lineages, and indicate the relative ages and order of appearance of those lineages. A common theme among these three unrelated clans of serine proteases is the development or maintenance of a catalytic tetrad, the fourth member of which is a Ser or Cys whose side chain helps stabilize other residues of the standard catalytic triad. A genetic mechanism for mutation of conserved markers, domain duplication followed by gene splitting, is suggested by analysis of evolutionary markers from newly sequenced genes with multiple protease domains.

A novel member of the subtilisin-like protease family from Bacillus subtilis.

aprX is a 1326 bp gene of Bacillus subtilis strain 168 that encodes a serine protease, probably intracellular, characterized by significant similarity with subtilisins, thermitases and pyrolysins. Transcription analysis, performed by RT-PCR and primer extension, allowed the localization of the active promoter and showed that aprX is expressed in stationary phase. The pattern of expression of aprX and its dependence on various transition state regulatory genes (degU, degQ, hpr, abrB, sinR), monitored by lacZ transcriptional fusions, are distinctive from those of subtilisin and other degradative enzymes. aprX is not essential for either growth or sporulation.