Structural and regulatory aspects of Src kinase activation

Kináza Src má stěžejní roli v množství fundamentálních buněčných procesů. Mimo jiné je součástí signálních drah řídících proliferaci, motilitu či diferenciaci a často bývá deregulována v různých typech nádorů. Aktivita Src proto podléhá přísné a komplexní regulaci, která je zprostředkována SH3 a SH2 doménami a fosforylačním stavem tyrosinů 416 a 527. V kompaktním inaktivním stavu kinázu udržují intramolekulárními inhibiční interakce. Jejich narušením dochází k otevření struktury Src a přechodu do aktivního stavu. Identifikovali jsme nový mechanismus, skrze který může být Src regulována. Jedná se o fosforylaci konzervovaného tyrosinu 90 vazebného povrchu SH3 domény, která vede ke snížení afinity SH3 domény k ligandům včetně CD linkeru a aktivaci kinázy. Fosfomimikujíci mutace tyrosinu 90 indukovala transformaci buněk a zvýšený invazivní potenciál. Jelikož je katalytická aktivita Src reflektována jeho strukturou, lze prostřednictvím stanovení tvaru kinázy usuzovat na její aktivitu. Na základě této korelace jsme sestrojili FRET senzor konformace Src umožňující sledovat s prostorovým a časovým rozlišením dynamiku aktivace kinázy v buňkách. Dokumentovali jsme, že aktivační mutace v SH3, SH2 i kinázové doméně nebo některé typy inhibitorů jsou schopny vyvolat otevření struktury Src. Analýza aktivace Src...Src kinase plays a crucial role in a multitude of fundamental cellular processes. Src is an essential component of signalling pathways controlling cellular proliferation, motility or differentiation, and is often found deregulated in tumours. Src activity is therefore maintained under stringent and complex regulation mediated by SH3 and SH2 domains and the phosphorylation state of tyrosines 416 and 527. Active Src adopts an open conformation whereas inactive state of the kinase is characterised by a compact structure stabilised by inhibitory intramolecular interactions. We identified phosphorylation of tyrosine 90 within binding surface of SH3 domain as a new regulatory switch controlling Src kinase activation. Using substitutions mimicking phosphorylation state of the residue we demonstrated that tyrosine 90 phosphorylation controls Src catalytic activity, conformation and interactions mediated by the SH3 domain, representing a positive regulatory mechanism leading to elevated activation of mitogenic pathways and increased invasive potential of cells. Based on correlation between compactness of Src structure and its catalytic activity, we constructed a FRET-based sensor of Src conformation enabling to measure the dynamics of Src activation in cells with spatio-temporal resolution. We found that...Department of Cell BiologyKatedra buněčné biologieFaculty of SciencePřírodovědecká fakult

Structural and regulatory aspects of Src kinase activation

Kináza Src má stěžejní roli v množství fundamentálních buněčných procesů. Mimo jiné je součástí signálních drah řídících proliferaci, motilitu či diferenciaci a často bývá deregulována v různých typech nádorů. Aktivita Src proto podléhá přísné a komplexní regulaci, která je zprostředkována SH3 a SH2 doménami a fosforylačním stavem tyrosinů 416 a 527. V kompaktním inaktivním stavu kinázu udržují intramolekulárními inhibiční interakce. Jejich narušením dochází k otevření struktury Src a přechodu do aktivního stavu. Identifikovali jsme nový mechanismus, skrze který může být Src regulována. Jedná se o fosforylaci konzervovaného tyrosinu 90 vazebného povrchu SH3 domény, která vede ke snížení afinity SH3 domény k ligandům včetně CD linkeru a aktivaci kinázy. Fosfomimikujíci mutace tyrosinu 90 indukovala transformaci buněk a zvýšený invazivní potenciál. Jelikož je katalytická aktivita Src reflektována jeho strukturou, lze prostřednictvím stanovení tvaru kinázy usuzovat na její aktivitu. Na základě této korelace jsme sestrojili FRET senzor konformace Src umožňující sledovat s prostorovým a časovým rozlišením dynamiku aktivace kinázy v buňkách. Dokumentovali jsme, že aktivační mutace v SH3, SH2 i kinázové doméně nebo některé typy inhibitorů jsou schopny vyvolat otevření struktury Src. Analýza aktivace Src...Src kinase plays a crucial role in a multitude of fundamental cellular processes. Src is an essential component of signalling pathways controlling cellular proliferation, motility or differentiation, and is often found deregulated in tumours. Src activity is therefore maintained under stringent and complex regulation mediated by SH3 and SH2 domains and the phosphorylation state of tyrosines 416 and 527. Active Src adopts an open conformation whereas inactive state of the kinase is characterised by a compact structure stabilised by inhibitory intramolecular interactions. We identified phosphorylation of tyrosine 90 within binding surface of SH3 domain as a new regulatory switch controlling Src kinase activation. Using substitutions mimicking phosphorylation state of the residue we demonstrated that tyrosine 90 phosphorylation controls Src catalytic activity, conformation and interactions mediated by the SH3 domain, representing a positive regulatory mechanism leading to elevated activation of mitogenic pathways and increased invasive potential of cells. Based on correlation between compactness of Src structure and its catalytic activity, we constructed a FRET-based sensor of Src conformation enabling to measure the dynamics of Src activation in cells with spatio-temporal resolution. We found that...Katedra buněčné biologieDepartment of Cell BiologyFaculty of SciencePřírodovědecká fakult

Biomarkers of Brain Damage: S100B and NSE Concentrations in Cerebrospinal Fluid-A Normative Study

NSE and S100B belong among the so-called structural proteins of the central nervous system (CNS). Lately, this group of structural proteins has been profusely used as specific biomarkers of CNS tissue damage. So far, the majority of the research papers have focused predominantly on the concentrations of these proteins in blood in relation to CNS damage of various origins. Considering the close anatomic and functional relationship between the brain or spinal cord and cerebrospinal fluid (CSF), in case of a CNS injury, a rapid and pronounced increase of the concentrations of structural proteins specifically in CSF takes place. This study inquires into the physiological concentrations of NSE and S100B proteins in CSF, carried out on a sufficiently large group of 601 patients. The detected values can be used for determination of a normal reference range in CSF in a clinical laboratory diagnostics

Structural and regulatory aspects of Src kinase activation

Src kinase plays a crucial role in a multitude of fundamental cellular processes. Src is an essential component of signalling pathways controlling cellular proliferation, motility or differentiation, and is often found deregulated in tumours. Src activity is therefore maintained under stringent and complex regulation mediated by SH3 and SH2 domains and the phosphorylation state of tyrosines 416 and 527. Active Src adopts an open conformation whereas inactive state of the kinase is characterised by a compact structure stabilised by inhibitory intramolecular interactions. We identified phosphorylation of tyrosine 90 within binding surface of SH3 domain as a new regulatory switch controlling Src kinase activation. Using substitutions mimicking phosphorylation state of the residue we demonstrated that tyrosine 90 phosphorylation controls Src catalytic activity, conformation and interactions mediated by the SH3 domain, representing a positive regulatory mechanism leading to elevated activation of mitogenic pathways and increased invasive potential of cells. Based on correlation between compactness of Src structure and its catalytic activity, we constructed a FRET-based sensor of Src conformation enabling to measure the dynamics of Src activation in cells with spatio-temporal resolution. We found that..

Physical education for pre-school children with autism spectrum disorder

3 Abstract Theme of thesis: Physical education for pre-school children with autism spectrum disorder Objectives of thesis: The aimt of my thesis was to prepare a methodical guide for teachers and trainers of free time activities for pre- school children with ASD, then to identify, if the bee can contribute to improve the children self-reliance during practice of their motorical skills, if it can have a positive effect on the behaviour of children during work and whether it can help reducing problematic behaviour. Method: The main metod used in the qualitative part of my research was observation participation method, the other informal interview. The data was evaluated using simple analysis and statistical method of frequencies. Results and conclusions: The objective of my thesis is to prepare a methodical guide for teachers and trainers of free time activities dealing with pre-school children with ASD. I found outied, that participation in a bee can contribute to improve the children's self-reliance during practice of their motorical skills, it also positively affected the behaviour of children during work and it helped to reduce problematic behaviour. Key words: Autistic spectrum disorder, physical education, structured teaching, structured movement activity, pre-school children, free time activit

Study of Glycine-NBDCl Derivate Stability

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of biophysics and physical chemistry Candidate: Lenka Koudelková Consultant: Ing.Vladimír Kubíček, CSc. Title of Thesis: Study of glycine-NBDCl derivate stability In this thesis the stability of glycine-NBDCl derivative under three different conditions (heat+light, heat+dark, cool+dark) is studied by HPLC with fluorimetric detection. Ater being stabilized by hydrochloric acid the derivatives exhibit a better stability, but the concentrations found during several weeks are too scattered. In addition an alternative chromatographic conditions were proposed for the HPLC analysis. Acetonitrile with hydrogen phosphate buffer (pH 7,0) in ratio 70:30 on an Atlantis column gave symetric peaks with a short time of elution

Structural and regulatory aspects of Src kinase activation

Src kinase plays a crucial role in a multitude of fundamental cellular processes. Src is an essential component of signalling pathways controlling cellular proliferation, motility or differentiation, and is often found deregulated in tumours. Src activity is therefore maintained under stringent and complex regulation mediated by SH3 and SH2 domains and the phosphorylation state of tyrosines 416 and 527. Active Src adopts an open conformation whereas inactive state of the kinase is characterised by a compact structure stabilised by inhibitory intramolecular interactions. We identified phosphorylation of tyrosine 90 within binding surface of SH3 domain as a new regulatory switch controlling Src kinase activation. Using substitutions mimicking phosphorylation state of the residue we demonstrated that tyrosine 90 phosphorylation controls Src catalytic activity, conformation and interactions mediated by the SH3 domain, representing a positive regulatory mechanism leading to elevated activation of mitogenic pathways and increased invasive potential of cells. Based on correlation between compactness of Src structure and its catalytic activity, we constructed a FRET-based sensor of Src conformation enabling to measure the dynamics of Src activation in cells with spatio-temporal resolution. We found that..

Structural and regulatory aspects of Src kinase activation

Src kinase plays a crucial role in a multitude of fundamental cellular processes. Src is an essential component of signalling pathways controlling cellular proliferation, motility or differentiation, and is often found deregulated in tumours. Src activity is therefore maintained under stringent and complex regulation mediated by SH3 and SH2 domains and the phosphorylation state of tyrosines 416 and 527. Active Src adopts an open conformation whereas inactive state of the kinase is characterised by a compact structure stabilised by inhibitory intramolecular interactions. We identified phosphorylation of tyrosine 90 within binding surface of SH3 domain as a new regulatory switch controlling Src kinase activation. Using substitutions mimicking phosphorylation state of the residue we demonstrated that tyrosine 90 phosphorylation controls Src catalytic activity, conformation and interactions mediated by the SH3 domain, representing a positive regulatory mechanism leading to elevated activation of mitogenic pathways and increased invasive potential of cells. Based on correlation between compactness of Src structure and its catalytic activity, we constructed a FRET-based sensor of Src conformation enabling to measure the dynamics of Src activation in cells with spatio-temporal resolution. We found that..

Abrasion resistance of hot-dip galvanization

V teoretické části této práce je popsán proces žárového zinkování a charakteristika zinkového povlaku. Dále jsou uvedeny vlivy některých prvků v oceli nebo v zinkové lázni na vlastnosti a strukturu vytvořeného povlaku. Experimentální část hodnotí odolnost proti otěru zinkového povlaku v závislosti na množství cínu, které obsahuje zinková lázeň.The theoretical part of this thesis describes process of hot-dip galvanizing and characteristics of zinc coating. There are also introduced effects of some elements in iron or in zinc bath on quality and structure of formed coating. The experimental part evaluates abrasion resistance of hot-dip galvanization depending on amount of tin contained in zinc bath

The role of tyrosine phosphorylation in hnRNA splicing

Kódující sekvence eukaryotických genů jsou přerušovány dlouhými úseky nekódující intronické DNA, která musí být po přepisu do heterogenní jaderné RNA z transkriptu vystřižena. Eukaryotní organismy se vzrůstajícím tlakem na komplexitu proteomu vyvinuly systém alternativního sestřihu, jež je schopen, prostřednictvím oslabených konsensus sekvencí sestřihových míst obklopených přídatnými regulačními RNA elementy a s nimi asociujícími sestřihovými faktory, vytvořit proteinové produkty podle aktuálních potřeb daných vnějšími a vnitřními vlivy. Síť kooperativně či antagonisticky působících faktorů, jež rozhoduje o rozeznání sestřihových míst, je regulována enzymatickými modifikacemi na základě aktivit odpovídajících signálních kaskád. Vyjma mnohých jiných enzymů zajišťuje tyto modulace i rodina protein tyrosin kináz. Připojení fosfátu na tyrosiny proteinů metabolismu RNA za katalýzy kinázové domény tak ovlivňuje jejich afinitu k RNA a dalším interakčním partnerům, lokalizaci, enzymatickou aktivitu či jiné vlastnosti, což vyústí v ustanovení nové interakční sítě a využití odlišných sestřihových míst. Vzniklé produkty potom mohou rozdílnou měrou inhibovat či spouštět nejrůznější buněčné procesy nebo jinak regulovat fyziologii organismů.Coding sequences of eukaryotic genes are interrupted by long segments of noncoding intronic DNA, which must be spliced after a transcription into a heterogenous nuclear RNA. Due to an increasing pressure on complexity of proteome eukaryotic organisms evolved alternative splicing. It is enabled through weak consensus sequences of splice sites flanked with accessory regulatory RNA elements, that associate with splicing factors, to create protein products according to current requirements implicated by outer and inner conditions. The net of cooperatively or antagonistic acting factors determines whether splice sites are recognized or not. This molecular system is regulated by enzymatic modifications depending on activity of corresponding signaling pathways. Beside many other enzymes a family of protein tyrosine kinases is involved in the process. Via catalytic activity of their kinase domains, they add phosphate to tyrosines of proteins that participate in RNA metabolism. Phosphorylation affects their affinity for RNA and other interacting partners, localization, enzymatic activity or other properties. The changes result in establishing of new setting of regulatory net and usage of distinct splice sites. Products then may with a different efficiency inhibit or trigger various cell processes or...Katedra buněčné biologieDepartment of Cell BiologyPřírodovědecká fakultaFaculty of Scienc