BACKGROUND: Serine proteases are important virulence factors for many pathogens. Recently, we discovered a group of trypsin-like serine proteases with domain organization unique to flatworm parasites and containing a thrombospondin type 1 repeat (TSR-1). These proteases are recognized as antigens during host infection and may prove useful as anthelminthic vaccines, however their molecular characteristics are under-studied. Here, we characterize the structural and proteolytic attributes of serine protease 2 (SmSP2) from Schistosoma mansoni, one of the major species responsible for the tropical infectious disease, schistosomiasis. METHODOLOGY/PRINCIPAL FINDINGS: SmSP2 comprises three domains: a histidine stretch, TSR-1 and a serine protease domain. The cleavage specificity of recombinant SmSP2 was determined using positional scanning and multiplex combinatorial libraries and the determinants of specificity were identified with 3D homology models, demonstrating a trypsin-like endopeptidase mode of action. SmSP2 displayed restricted proteolysis on protein substrates. It activated tissue plasminogen activator and plasminogen as key components of the fibrinolytic system, and released the vasoregulatory peptide, kinin, from kininogen. SmSP2 was detected in the surface tegument, esophageal glands and reproductive organs of the adult parasite by immunofluorescence microscopy, and in the excretory/secretory products by immunoblotting. CONCLUSIONS/SIGNIFICANCE: The data suggest that SmSP2 is secreted, functions at the host-parasite interface and contributes to the survival of the parasite by manipulating host vasodilatation and fibrinolysis. SmSP2 may be, therefore, a potential target for anti-schistosomal therapy.
- MeSH
- fibrinolýza účinky léků MeSH
- hemokoagulace účinky léků MeSH
- hemostatika antagonisté a inhibitory MeSH
- krevní tlak účinky léků MeSH
- molekulární modely MeSH
- plazminogen účinky léků MeSH
- proteinové domény MeSH
- proteiny červů chemie genetika farmakologie MeSH
- proteolýza účinky léků MeSH
- rekombinantní proteiny MeSH
- Schistosoma mansoni enzymologie MeSH
- schistosomiasis mansoni parazitologie MeSH
- sekvence aminokyselin MeSH
- serinové endopeptidasy chemie genetika farmakologie MeSH
- tkáňový aktivátor plazminogenu účinky léků MeSH
- vazodilatace účinky léků MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Pepsin-family aspartic peptidases are biosynthesized as inactive zymogens in which the propeptide blocks the active site until its proteolytic removal upon enzyme activation. Here, we describe a novel dual regulatory function for the propeptide using a set of crystal structures of the parasite cathepsin D IrCD1. In the IrCD1 zymogen, intramolecular autoinhibition by the intact propeptide is mediated by an evolutionarily conserved exosite on the enzyme core. After activation, the mature enzyme employs the same exosite to rebind a small fragment derived from the cleaved propeptide. This fragment functions as an effective natural inhibitor of mature IrCD1 that operates in a pH-dependent manner through a unique allosteric inhibition mechanism. The study uncovers the propeptide-binding exosite as a target for the regulation of pepsin-family aspartic peptidases and defines the structural requirements for exosite inhibition.
- MeSH
- aktivace enzymů MeSH
- alosterická regulace MeSH
- katalytická doména MeSH
- kathepsin D chemie metabolismus MeSH
- kinetika MeSH
- klíšťata enzymologie MeSH
- koncentrace vodíkových iontů MeSH
- krystalografie rentgenová MeSH
- ligandy MeSH
- peptidy chemie metabolismus MeSH
- prekurzory enzymů chemie metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Mezi hlavní zátěžové situace a faktory působící na ošetřovatelský personál v oblasti psychické i fyzické figuruje péče a kontakt s umírajícími, nevyléčitelnými a trpícími pacienty, agresivními pacienty, zmatenými pacienty, rodinami pacientů a dále pracovní a osobní podmínky. V neposlední řadě také působení konfliktu (střetu) názorů, neshod a rozporu v morální oblasti. K poskytování efektivní péče v těchto situacích je nutné, aby pečující osoby byly sociálně zralé a vybaveny účinnými komunikačními dovednostmi. Ty také mohou usnadnit zvládání prožívání zátěžových situací ošetřovatelského personálu. Proto bylo naše výzkumné šetření zaměřeno na zmapování způsobů, kterými se ošetřovatelský personál vyrovnává se zátěžovými situacemi v rámci své profese a na rozdíly ve vyrovnání se s těmito situacemi ve vztahu k oddělení, kde pracuje. Metodika: Výzkumné šetření bylo realizováno kvantitativní metodou technikou dotazníku vlastní konstrukce v únoru až červnu 2016. Obsahoval 20 otázek zaměřených na zátěžové situace, jejich zvládání a podporu managementu v jednotlivých zdravotnických zařízeních. K analýze bylo použito systémů Microsoft Excel 2010 a Software R ve verzi 3.0.2 (Chí kvadrát test, Fisherův exaktní test). Celkem bylo rozdáno 300 dotazníků pro ošetřovatelský personál ve 4 nemocnicích a 3 kamenných hospicech a 7 zařízeních domácí hospicové péče. Návratnost byla 269 (90 %). Ošetřovatelský personál pociťuje zátěžové situace velmi často. Nejvíce zatěžující je pro respondenty z hospiců trpící pacient a pro respondenty z nemocnic převažuje kolektiv a péče o agresivního pacienta. Pro vyrovnávání se s pracovní zátěží se nejvíce osvědčily relaxace, odpočinek a spánek. Podporu v zátěžových situacích od zaměstnavatele pociťuje minimum respondentů, i když určitá podpora je poskytována formou školení, supervize či příspěvku na „zotavenou“. Respondenti by však více jako podporu od managementu uvítali např. zvýšení platu či více volna. Doporučovali by především různá školení na téma stres a jeho zvládání.
This paper deals with the stressful situations in the profession of the nursing staff. The occupation of the nurse belongs to the most demanding ones. In this occupation, the nurse is permanently affected by many stressors of different types. She faces death, the suffering, dying and incurably ill people very often. She is there in times of pain, agony and suffering. Personal encounters with these difficult situations require highly professional approach but most of all, they present immense work stress for the nurse. The stressful situations that are the most common and most difficult in the work of the nursing staff are the care of dyeing persons, the care of incurable and suffering persons, also the care of aggressive patients and confused patients. Patient’s family members could cause the stressful situations for nursing staff so as the working and personal conditions of nursing staff. The emotions the nursing personal experiences in such situation influence their behaviour and communication with patients in negative way. Both sides are therefore dissatisfied which could develop in to aggressive situation or burn out syndrome of the personnel. Also such culminate situations could lead to leaving the personnel. The lack of personnel then doesn´t allows enough of needed time for communication with patients in their life difficult situations. The first objective of our survey was to map the manners used by the nursing personnel to cope with the stressful situations brought by their profession. The second objective was to find out the difference in the coping with the stressful situations between the hospital staff and hospice staff. The research section of this survey contains the quantitative research was done by using the technique of an anonymous questionnaire with 20 question prepared by ourselves. The questionnaire was distributed to the nursing staff of all categories in the hospital and hospice care. It was completed by 269 respondents in total. The data was statistically processed using systems Microsoft Excel 2010 and Software R, version 3.0.2 (Chi-squared test, Fisher´s exact test). The research results show that the nursing staff faces the stressful situations very often. They see as the heaviest stress the care of the suffering and aggressive patient, conflicts with the patient´s family, conflicts at work and workload. The manners of coping with the work burden which proved as the best for them are relax, rest and sleep. There were no differences proved in the manners of coping with the stress among respective members of the nursing team. Nevertheless, the coping with the work burden of the nursing staff in the hospital and hospice care is different. A minimum of the respondents notices any support during the stressful situations from the employer. On the other hand, certain support by the management could be viewed in the form of trainings, supervision or contribution to a „recovery“ which are granted to the respondents from the employer according to their answers. However, the respondents would prefer, as the support by the management, the increase in pay or more days off. The respondents view as insufficient the preparation of the graduates for the future stressful situations in their profession, as well. They would recommend mainly various trainings dealing with the stress and its management. We can state that in the transformation and process of improvement of health care service in Czech Republic the nursing personnel in at the last place. The care of caregivers is insufficient the meaning of supervision, support and education of the personnel. A good solution to alleviate the work burden of the nursing staff and quality enhancement of the services provided could be investments into better work conditions, both in terms of sufficient human and material resources so as the very organization of the work processes. This means the investment into safety of patients because continuous stress in work could jeopardize the quality of provided service and care but also the health of providers of care.
- MeSH
- dospělí MeSH
- hospicová a paliativní ošetřovatelská péče pracovní síly MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- péče o dospělé pacienty metody organizace a řízení pracovní síly MeSH
- personál sesterský nemocniční psychologie MeSH
- pracovní zátěž klasifikace psychologie MeSH
- průzkumy a dotazníky MeSH
- psychický stres etiologie prevence a kontrola psychologie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
Cathepsin B1 (SmCB1) is a digestive protease of the parasitic blood fluke Schistosoma mansoni and a drug target for the treatment of schistosomiasis, a disease that afflicts over 200 million people. SmCB1 is synthesized as an inactive zymogen in which the N-terminal propeptide blocks the active site. We investigated the activation of the zymogen by which the propeptide is proteolytically removed and its regulation by sulfated polysaccharides (SPs). We determined crystal structures of three molecular forms of SmCB1 along the activation pathway: the zymogen, an activation intermediate with a partially cleaved propeptide, and the mature enzyme. We demonstrate that SPs are essential for the autocatalytic activation of SmCB1, as they interact with a specific heparin-binding domain in the propeptide. An alternative activation route is mediated by an S. mansoni asparaginyl endopeptidase (legumain) which is downregulated by SPs, indicating that SPs act as a molecular switch between both activation mechanisms.
BACKGROUND: Enzymatic allergens of storage mites that contaminate stored food products are poorly characterized. We describe biochemical and immunological properties of the native alpha-amylase allergen Aca s 4 from Acarus siro, a medically important storage mite. RESULTS: A. siro produced a high level of alpha-amylase activity attributed to Aca s 4. This enzyme was purified and identified by protein sequencing and LC-MS/MS analysis. Aca s 4 showed a distinct inhibition pattern and an unusual alpha-amylolytic activity with low sensitivity to activation by chloride ions. Homology modeling of Aca s 4 revealed a structural change in the chloride-binding site that may account for this activation pattern. Aca s 4 was recognized by IgE from house dust mite-sensitive patients, and potential epitopes for cross-reactivity with house dust mite group 4 allergens were found. CONCLUSIONS: We present the first protein-level characterization of a group 4 allergen from storage mites. Due to its high production and IgE reactivity, Aca s 4 is potentially relevant to allergic hypersensitivity.
- MeSH
- Acaridae enzymologie imunologie MeSH
- alergeny chemie imunologie izolace a purifikace MeSH
- alergie krev imunologie MeSH
- alfa-amylasy chemie imunologie izolace a purifikace MeSH
- feces chemie MeSH
- hmyzí proteiny chemie imunologie izolace a purifikace MeSH
- imunoglobulin E krev MeSH
- lidé MeSH
- molekulární sekvence - údaje MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- strukturní homologie proteinů MeSH
- terciární struktura proteinů MeSH
- vazba proteinů MeSH
- zkřížené reakce MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Blood flukes of the genus Schistosoma cause the disease schistosomiasis that infects over 200 million people worldwide. Treatment relies on just one drug, and new therapies are needed should drug resistance emerge. Schistosoma mansoni cathepsin B1 (SmCB1) is a gut-associated protease that digests host blood proteins as source of nutrients. It is under evaluation as a therapeutic target. Enzymatic activity of the SmCB1 zymogen is prevented by the pro-peptide that sterically blocks the active site until activation of the zymogen to the mature enzyme. We investigated the structure-inhibition relationships of how the SmCB1 pro-peptide interacts with the enzyme core using a SmCB1 zymogen model and pro-peptide-derived synthetic fragments. Two regions were identified within the pro-peptide that govern its inhibitory interaction with the enzyme core: an "active site region" and a unique "heparin-binding region" that requires heparin. The latter region is apparently only found in the pro-peptides of cathepsins B associated with the gut of trematode parasites. Finally, using the active site region as a template and a docking model of SmCB1, we designed a series of inhibitors mimicking the pro-peptide structure, the best of which yielded low micromolar inhibition constants. Overall, we identify a novel glycosaminoglycan-mediated mechanism of inhibition by the pro-peptide that potentially regulates zymogen activation and describe a promising design strategy to develop antischistosomal drugs.
- MeSH
- chromatografie afinitní MeSH
- heparin farmakologie MeSH
- inhibitory proteinkinas chemická syntéza chemie farmakologie MeSH
- katalytická doména účinky léků MeSH
- kathepsin B antagonisté a inhibitory metabolismus MeSH
- molekulární modely MeSH
- molekulární struktura MeSH
- peptidové mapování MeSH
- peptidy chemie farmakologie MeSH
- prasata MeSH
- racionální návrh léčiv MeSH
- Schistosoma mansoni enzymologie MeSH
- sekundární struktura proteinů MeSH
- střevní sliznice chemie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Propeptide blocks the active site in the inactive zymogen of cathepsin D and is cleaved off during zymogen activation. We have designed a set of peptidic fragments derived from the propeptide structure and evaluated their inhibitory potency against mature cathepsin D using a kinetic assay. Our mapping of the cathepsin D propeptide indicated two domains in the propeptide involved in the inhibitory interaction with the enzyme core: the active site "anchor" domain and the N-terminus of the propeptide. The latter plays a dominant role in propeptide inhibition (nanomolar Ki), and its high-affinity binding was corroborated by fluorescence polarization measurements. In addition to the inhibitory domains of propeptide, a fragment derived from the N-terminus of mature cathepsin D displayed inhibition. This finding supports its proposed regulatory function. The interaction mechanisms of the identified inhibitory domains were characterized by determining their modes of inhibition as well as by spatial modeling of the propeptide in the zymogen molecule. The inhibitory interaction of the N-terminal propeptide domain was abolished in the presence of sulfated polysaccharides, which interact with basic propeptide residues. The inhibitory potency of the active site anchor domain was affected by the Ala38pVal substitution, a propeptide polymorphism reported to be associated with the pathology of Alzheimer's disease. We infer that propeptide is a sensitive tethered ligand that allows for complex modulation of cathepsin D zymogen activation.
- MeSH
- aminokyselinové motivy MeSH
- financování organizované MeSH
- glykosaminoglykany metabolismus MeSH
- inhibitory proteas chemická syntéza metabolismus MeSH
- katalytická doména MeSH
- kathepsin D antagonisté a inhibitory chemie metabolismus MeSH
- lidé MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- peptidové fragmenty antagonisté a inhibitory chemická syntéza metabolismus MeSH
- peptidové mapování MeSH
- prekurzory enzymů antagonisté a inhibitory chemie metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
