Mechanismus působení nepeptidových inhibitorů HIV proteasy

Inhibice HIV-1 proteasy hraje významnou roli v boji proti viru HIV. Od roku 1995 bylo na trh uvedeno devět inhibitorů HIV-1 proteasy, avšak jejich účinnost klesá vinou vzniku rezistence viru vůči inhibitorům. Proto je potřeba vyvíjet účinnější léčiva s novými strukturními motivy a mechanismy účinku. V nedávné době byla u několika inhibitorů pozorována jejich vazba i mimo aktivní místo proteasy. Darunavir, jenž je posledním schváleným inhibitorem HIV-1 proteasy a vykazuje kompetitivní typ inhibice, se pravděpodobně také váže do oblasti chlopní HIV-1 proteasy. Dvě studie podporující tento alternativní způsob vazby byly založeny na kinetických měřeních a rentgenové krystalografii. Je však otázkou, zda-li dochází k této vazbě i za fyziologických podmínek anebo zda-li se jedná pouze o krystalizační artefakt. I další data podporují alternativní vazbu darunaviru s vysoce mutovanou HIV-1 proteasou: pomocí termodynamické analýzy bylo prokázano, že na dimerní HIV-1 proteasu se váží dvě molekuly darunaviru. Nicméně toto pozorování nebylo potvrzeno rentgenovou krystalografií, neboť darunavir byl v krystalové struktuře přítomen pouze v aktivním místě HIV-1 proteasy. Tato vysoce mutovaná varianta HIV-1 proteasy se proto stala předmětem dalšího zkoumání. Pro studium mechanismu vazby darunaviru na mutovanou...The inhibition of HIV-1 protease plays an important role in combating HIV. Nine HIV-1 protease inhibitors have been succesfully marketed for the treatment since 1995. However, their efficiencies decrease due to the resistance development. More potent compounds with novel structural motifs and mechanisms of action are therefore still needed. Several inhibitory compounds have been reported to bind to the protease at the loci different from the active site. Interestingly, darunavir, which is the last approved inhibitor with supposedly competitive mode of action, was also suggested to bind to the flap region of the protease. Two studies discussed this alternative binding mode based on the X-ray structural and kinetic analysis, respectively. Nevertheless, it is questionable, if such a mechanism is relevant also in physiological conditions or if it is only an artifact of crystallization. Another study provided a strong evidence for the alternative binding of darunavir to highly mutated HIV-1 protease. Based on thermodynamic analysis, it was shown that two molecules of darunavir bind to the protease dimer. Surprisingly, this observation was not confirmed by the X-ray structure analysis since the inhibitor was bound only within the active site. However, this protease variant was employed in further...Katedra biochemieDepartment of BiochemistryPřírodovědecká fakultaFaculty of Scienc

Intramembránové proteasy z rodiny rhomboidů v prokaryotech: mechanismus, repertoáry substrátů a biologické funkce u Gram-positivní bakterie Bacillus subtilis.

(Czech) Proteázy z rodiny rhomboidů patří do rozsáhlé skupiny serinových intramembránových proteáz, které katalyzují proteolytické štěpení membránových proteinů uvnitř jejich transmembránových oblastí, v hydrofobním prostředí lipidických buněčných membrán. Rhomboidové proteázy byly objeveny v roce 2001 v Drosophila. V průkopnické studii (Lee et al.) byla identifikována zásadní role Rhomboidu-1 (Rhom-1), který v rané fázi vývoje oka octomilky aktivuje signální dráhu receptoru epidermálního růstového faktoru. Rhomboidové proteázy, ať už aktivní proteázy (rhomboidy) či jejich katalyticky neaktivní protějšky (rhomboidové proteiny zahrnující iRhomy a Derliny), jsou silně konzervovány, což naznačuje jejich biologickou významnost. Rhomboidy jsou přítomné napříč živočišnými říšemi, od archea po člověka, zatímco proteolyticky neaktivní rhomboidové proteiny jsou přítomné výlučně v eukaryotních organizmech. Rodina rhomboidových proteinů hraje roli v široké škále rozmanitých biologických procesů, jakými je signalizace ve vývoji metazoí, mitochondriální biogenezi kvasinek, invaze protozoálních parazitů do hostitelských buněk, kvalitativní kontrola proteinů v endoplazmatickém retikulu (ER), či bakteriální quorum sensing. Ze strukturního i mechanistického hlediska jsou rhomboidy nejprostudovanějšími z...Rhomboid proteases are a class of serine intramembrane proteases, a large family of enzymes that catalyze the proteolytic cleavage of membrane proteins within their transmembrane regions, in the hydrophobic environment of cellular lipid membranes. Rhomboid proteases were discovered in 2001 in Drosophila. In their pioneering study, Lee et al. identified the essential role of Rhomboid-1 protein (Rhom-1), which proteolytically activates the epidermal growth factor (EGF) receptor signaling pathway, in the early stages of fly eye development. Members of the rhomboid superfamily - active proteases (rhomboids) as well as their catalytically-dead counterparts (rhomboid-like proteins, including iRhoms and Derlins) - are widely conserved, implying their biological significance. Rhomboids are present in all kingdoms of life from archea to humans, while proteolytically inactive rhomboid-like proteins are present in eukaryotes only. Rhomboid superfamily proteins play roles in a wide range of processes, as diverse as signaling in metazoan development, mitochondrial biogenesis in yeast, host- cell invasion by protozoan parasites, protein quality control in the endoplasmic reticulum (ER) or bacterial quorum sensing. Rhomboids are the best understood intramembrane proteases from a structural and mechanistic points...Katedra genetiky a mikrobiologieDepartment of Genetics and MicrobiologyFaculty of SciencePřírodovědecká fakult

Grid and Cloud Computing

The bachelor thesis deals with cloud and grid technologies. The first part discusses cloud computing technology, its components, infrastructure, models, advantages, disadvantages, and security risks. The following section describes computing and data grids, their function, use and security. The last part of the thesis shows an overview of the commercial market of cloud and grid services

Rhomboid family intramembrane proteases in prokaryotes: mechanism, substrate repertoires and biological functions in the Gram-positive bacterium Bacillus subtilis.

Rhomboid proteases are a class of serine intramembrane proteases, a large family of enzymes that catalyze the proteolytic cleavage of membrane proteins within their transmembrane regions, in the hydrophobic environment of cellular lipid membranes. Rhomboid proteases were discovered in 2001 in Drosophila. In their pioneering study, Lee et al. identified the essential role of Rhomboid-1 protein (Rhom-1), which proteolytically activates the epidermal growth factor (EGF) receptor signaling pathway, in the early stages of fly eye development. Members of the rhomboid superfamily - active proteases (rhomboids) as well as their catalytically-dead counterparts (rhomboid-like proteins, including iRhoms and Derlins) - are widely conserved, implying their biological significance. Rhomboids are present in all kingdoms of life from archea to humans, while proteolytically inactive rhomboid-like proteins are present in eukaryotes only. Rhomboid superfamily proteins play roles in a wide range of processes, as diverse as signaling in metazoan development, mitochondrial biogenesis in yeast, host- cell invasion by protozoan parasites, protein quality control in the endoplasmic reticulum (ER) or bacterial quorum sensing. Rhomboids are the best understood intramembrane proteases from a structural and mechanistic points..

Kinetic, thermodynamic and structural characterisation of mutated HIV-1 proteases resistant towards inhibitors

Department of BiochemistryKatedra biochemieFaculty of SciencePřírodovědecká fakult

Grid and Cloud Computing

Bakalárska práca pojednáva o technológiách cloudov a gridov. Prvá časť práce rozoberá technológiu cloud computingu, jeho komponenty, infraštruktúru, modely, výhody, nevýhody a bezpečnostné riziká. Nasledujúca časť popisuje výpočtové a dátové gridy, ich funkciu, využitie a bezpečnosť. Posledná časť práce zobrazuje prehľad komerčného trhu cloudových a gridových služieb.The bachelor thesis deals with cloud and grid technologies. The first part discusses cloud computing technology, its components, infrastructure, models, advantages, disadvantages, and security risks. The following section describes computing and data grids, their function, use and security. The last part of the thesis shows an overview of the commercial market of cloud and grid services.

Rhomboid family intramembrane proteases in prokaryotes: mechanism, substrate repertoires and biological functions in the Gram-positive bacterium Bacillus subtilis.

Rhomboid proteases are a class of serine intramembrane proteases, a large family of enzymes that catalyze the proteolytic cleavage of membrane proteins within their transmembrane regions, in the hydrophobic environment of cellular lipid membranes. Rhomboid proteases were discovered in 2001 in Drosophila. In their pioneering study, Lee et al. identified the essential role of Rhomboid-1 protein (Rhom-1), which proteolytically activates the epidermal growth factor (EGF) receptor signaling pathway, in the early stages of fly eye development. Members of the rhomboid superfamily - active proteases (rhomboids) as well as their catalytically-dead counterparts (rhomboid-like proteins, including iRhoms and Derlins) - are widely conserved, implying their biological significance. Rhomboids are present in all kingdoms of life from archea to humans, while proteolytically inactive rhomboid-like proteins are present in eukaryotes only. Rhomboid superfamily proteins play roles in a wide range of processes, as diverse as signaling in metazoan development, mitochondrial biogenesis in yeast, host- cell invasion by protozoan parasites, protein quality control in the endoplasmic reticulum (ER) or bacterial quorum sensing. Rhomboids are the best understood intramembrane proteases from a structural and mechanistic points..

Mechanism of action of non-peptide inhibitors of HIV protease

The inhibition of HIV-1 protease plays an important role in combating HIV. Nine HIV-1 protease inhibitors have been succesfully marketed for the treatment since 1995. However, their efficiencies decrease due to the resistance development. More potent compounds with novel structural motifs and mechanisms of action are therefore still needed. Several inhibitory compounds have been reported to bind to the protease at the loci different from the active site. Interestingly, darunavir, which is the last approved inhibitor with supposedly competitive mode of action, was also suggested to bind to the flap region of the protease. Two studies discussed this alternative binding mode based on the X-ray structural and kinetic analysis, respectively. Nevertheless, it is questionable, if such a mechanism is relevant also in physiological conditions or if it is only an artifact of crystallization. Another study provided a strong evidence for the alternative binding of darunavir to highly mutated HIV-1 protease. Based on thermodynamic analysis, it was shown that two molecules of darunavir bind to the protease dimer. Surprisingly, this observation was not confirmed by the X-ray structure analysis since the inhibitor was bound only within the active site. However, this protease variant was employed in further..