Aplikovaná chemie a biochemie Přednáška č. 2 Proteinové techniky (1) Proteinové techniky (1): ˇlokalizace v buňce (live cell imaging; imunofluorescenční techniky; frakcionace); * interakce mezi proteiny (imunoprecipitace; GST-pull-down; yeast two-hybrid screen); * interakce s DNA ; detekce transkripční aktivity Lokalizace proteinu v buňce * imunodetekce ­ imunofluorescenční a imunohistochemické techniky; kolokalizace proteinů ­ konfokální mikroskopie; Marker dané organely (subcel. komp.), negativní kontrola. Enzymové (HRP, AP) immunofluorescenční. techniky. Hlavní výhody jsou senzitivita možnost vícenásobného barvení, nevýhodou je fading, autofluorescence a náročné technické vybavení. Nevýhodou imunohistochemických technik je také vznik artefaktů v důsledku fixace buněk. AhR ve varlatech potkana: sekundární protilátka značená + TCDD; 30 min Příklad: Lokalizace Ah receptoru v buňce AhR ve buněčné linii WB-F344: sekundární protilátka značená FITC Lokalizace v buňce * live cell imaging ­ využití proteinů značených např. fluorescenčními proteiny ­ time-lapse video, FLIP, FRET; Výhoda ­ možnost pracovat s živými buňkami ­ je možné sledovat translokace proteinů, pohyby organel, pohyb proteinů v rámci buňky, stanovit přibližné relativní množství proteinu i proteinové interakce (intermolecular FRET). Fluorescenční markery: GFP Fluorescence can be used to visualize cell structures at many levels. Originally, fluorescence was mainly observed from small organic dyes attached by means of antibodies to the protein of interest. However, antibody targeting of intracellular proteins normally requires cell fixation and permeabilization. Later, fluorophores could directly recognize organelles, nucleic acids, and certain important ions in living cells. In the past decade, fluorescent proteins have enabled noninvasive imaging in living cells and organisms of reporter gene expression, protein trafficking, and many dynamic biochemical signals. Fluorescenční markery: Fluorescenční markery: Fluorescenční markery: Lokalizace v buňce * subcelulární frakcionace následovaná WB Výhoda ­ nižší spotřeba protilátek, snadné vyloučení nespecifických vazeb protilátek; nevýhoda ­ pracnost. Nejčastěji používaná metoda ­ použití specifických detergentů, ultracentrifugace. Sequential centrifugation: Nuclear fraction - at 1,000g for 10 min. Heavy mitochondrial fraction 3,000g for 10min. Light mitochondrial fraction at 15,000-17,000g for 10 min. Microsomal fraction at 100,000g for 45 min. Composition of the pellets The Nuclear Pellet contains, in addition to nuclei, mitochondria, sheets of plasma membrane (if present) and, if the homogenate has not been filtered, unbroken cells and debris (including connective tissue). Formation of this pellet is sometimes carried out at 500g rather than 1000g. The Heavy Mitochondrial Pellet contains predominantly, mitochondria with rathe few contaminants and is a common source of these organelles for respiratory studies. Minor components such as lysosomes, peroxisomes, Golgi membranes and various membrane vesicles are present largely because of entrapment during the pelleting process. Some plasma membrane fragments may also be present. These contaminants can be reduced by repeated washing. The Light Mitochondrial Pellet contains mitochondria, lysosomes, peroxisomes, Golgi membranes and some endoplasmic reticulum. Of all differential centrifugation fractions it is the most variable in terms of the actual centrifugation parameters used: g-forces of 15-20,000g and times of 10-20 min are the most common. Some methods are designed to maintain the Golgi membranes in their "stacked" form so that they sediment at much lower g-forces (see ref 3 for more information) The Microsomal Pellet is rather better defined and contains only membrane vesicles. Some of those vesicles will have been present in the cell before homogenization (e.g. endosomes, secretory vesicles and vesicles from the transGolgi network), others from the plasma membrane, Golgi and smooth and rough endoplasmic reticulum, will have been produced by the homogenization procedure. Characterization of DR depletion in Hepa1c1c7 cells. A, TCDD (10 nM) was added to Hepa1c1c7 cells for the times indicated before whole cell protein extracts were separated using 8% SDS-PAGE and analyzed by immunoblotting with anti-DR mAb. B, TCDD (10 nM) and/or Me2SO vehicle were added to Hepa1c1c7 cells for 2.5 h. MG132 (2.5 mM), E64 (10 mM), or vehicle (DMSO) were added at the same time in the lanes shown. Cytosolic and nuclear extracts were prepared and analyzed by 8% SDS-PAGE and immunoblotting using anti-DR mAb. Interakce mezi proteiny * stabilní ­ proteiny vytvářejí komplex * tranzientní ­ např. interakce enzymu s proteinovým substrátem * afinita dvou proteinů je dána rovnovážnou disociační konstantou ­ Kd = [A]*[B]/[AB] Základní metody: * GST pull-down assay * two-hybrid methods * co-immunoprecipitation * FRET GST pull-down assay is a simple technique to test interaction between a tagged protein or the bait (GST, His6, biotin ...) and another protein (test protein, or prey). The bait protein, purified from an appropriate expression system (e.g., Escherichia coli), is immobilized on a glutathione affinity gel. The bait serves as the secondary affinity support for identifying new protein partners or for confirming a previously suspected protein partner to the bait. Prey protein can be obtained from multiple sources including recombinant purified proteins, cell lysate or in vitro transcription/translation reactions. Protein-protein interactions can be visualized by SDS-PAGE and associated detection methods depending on the sensitivity requirements of the interacting proteins. These methods include Coomassie or silver staining, Western blotting and [35S] radioisotopic detection. GST pull-down assay Two-hybrid assays (interaction trap): Two chimeras, one containing the DNA-binding domain and one that contains an activation domain, are cotransfected into an appropriate host strain. If the fusion partners (yellow and red) interact, the DB and AD are brought into proximity and can activate transcription of reporter gene. Two-hybrid assay (interaction trap): * používají se hostitelské kmeny kvasinek (např. EGY48, EGY191) exprimující reportérový gen a dále gen pro rezistenci, např. LEU2, který umožňuje růst na médiu bez obsahu Leu; oba geny jsou pod kontrolou operátoru LexA * prvním krokem je příprava ,,bait" proteinu fúzovaného s LexA a transformace kvasinek tímto konstruktem; * selekce kmenů, ve kterých bait protein nepůsobí jako aktivátor, ani jako represor transkripce reportérového genu; * příprava cDNA knihovny, nebo specifických proteinů fúzovaných s aktivační doménou (B42 pro LEU2 a LacZ); Point mutations in the LXCXE motif disrupt the AhR-pRb interaction. Site-directed mutations were introduced into the AhR to substitute each of the conserved residues in the LXCXE motif with an alanine. Yeast cells containing a LacZ reporter plasmid (p8op-LacZ) were transformed with the wild-type (WT) or various alanine-substituted AhR bait constructs (L331A, C333A, E335A) in the absence (+pRb) and presence (-pRb) of plasmid pB42ADpRb and grown on leucine-deficient X-gal plates at 30°C for 3 to 5 days. [35S]AHR was synthesized in vitro in the presence (T) or absence (0) of 100 nM TCDD. Aliquots of the unfractionated translation products (35SAHR) or of two successive effluents from control nickel-agarose columns (Cont-1 and Cont-2) or RB-bearing columns (pRB-1 and pRB-2) were analyzed by SDS-PAGE. Protein-protein interactions between AHR and RB. Aliquots of 1 mg each of whole cell lysates were incubated with monospecific rabbit anti-RB antiserum, anti-AHR antiserum, or control rabbit polyclonal serum. Bound proteins were recovered with protein A plus protein G-Sepharose, washed, eluted by boiling, and analyzed by SDS-PAGE and immunoblotted with anti-RB and anti AHR antibodies SRC-1 mutants were fused to glutathione S-transferase and expressed in Escherichia coli. Expressed fusion proteins from bacterial cell lysates were put on glutathione-agarose beads. Protein coupled to beads was incubated with in vitro-translated [35S]methionine-labeled ARNT or RIN1, and absorbed proteins were subjected to SDS-PAGE. The two left lanes contained 1 l of in vitro-translated RIN1 and 1 l of in vitro-translated mARNT (25% input) for comparison. FRET FRET ­ příklad: Analysis of ligandinduced YFP-ER-CFPER interactions in MCF- 7 cells. A, representative FRET images. Images were acquired in COS-1 cells transfected with YFP-ER and CFP-ER. Interakce proteinu s DNA * příprava cytosolového nebo jaderného extraktu; * electrophoretic mobility shift assay ­ EMSA (gel retardation assay); * methylation interference; DNAse I protection assay ,,footprinting" EMSA * příprava značené DNA próby (end labeling, nick translation) * binding reaction (jaderný nebo cytosolový extrakt, carrier DNA a značené DNA próby) loading buffer * nedenaturující PAGE * vysušení gelu a autoradiografie EMSA EMSA Kontrola specifity DNase I foot printing analysis A DNA fragment (117 bp for the coding strand and 112 bp for the non-coding strand) containing a single copy of HRE or XRE in the central part and incubated with DNase I. The digeste DNA was precipitated with ethanol and separated in the denaturating acrylamide gel containing 8 M urea. Identifikace sekvencí, na které se vážou proteiny: * např. XRE (DRE) - N T/G T GCGTG A/C G/T A/T A/G N * genome-wide analysis ! Řada těchto sekvencí neaktivuje transkripci ! Reporter gene assays * stabilní * transientní; kontrola ,,transfection efficiency" (Renilla luciferase, GFP, -gal) * důležité vlastnosti: * hladina proteinu = hladina mRNA * snadno detekovatelný produkt * nesmí se nacházet v cílové buňce * nesmí ovlivňovat cílovou buňku Plasmid vectors Reportérové geny: GFP reporter Luciferase reporter * možnost sledovat dynamiku odpovědi -Galactosidase reporter - možnost využití k in vivo experimentům; Expression domains of Hox genes in a mouse. The photographs show whole embryos displaying the expression domains of two genes of the HoxB complex (blue stain). These domains can be revealed by in situ hybridization or, as in these examples, by constructing transgenic mice containing the control sequence of a Hox gene coupled to a LacZ reporter gene, whose product is detected histochemically. Each gene is expressed in a long expanse of tissue with a sharply defined anterior limit. The earlier the position of the gene in its chromosomal complex, the more anterior the anatomical limit of its expression. Thus, with minor exceptions, the anatomical domains of the successive genes form a nested set, ordered according to the ordering of the genes in the chromosomal complex. Thin layer chromatography ­ + + CAT reporter * nevýhodou je radioaktivní detekce; často pracná Proteiny (2): * funkce jednotlivých domén; * posttranslační modifikace (fosforylace, acetylace, glykosylace); * regulace odborávání proteinu (ubikvitinace; proteazomální degradace); * manipulace s proteinem (overexprese; dominant negative constructs, antisense oligonucleotides, siRNA).