protein localization
Dotaz
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Practical approach series
1st ed. xxiv, 231 s.
- Klíčová slova
- imunofluorescence,
- MeSH
- fluorescence MeSH
- fluorescenční mikroskopie MeSH
- proteiny MeSH
- Publikační typ
- příručky MeSH
- Konspekt
- Biochemie. Molekulární biologie. Biofyzika
- NLK Obory
- chemie, klinická chemie
- biochemie
Caspase-2 is an apical protease responsible for the proteolysis of cellular substrates directly involved in mediating apoptotic signaling cascades. Caspase-2 activation is inhibited by phosphorylation followed by binding to the scaffolding protein 14-3-3, which recognizes two phosphoserines located in the linker between the caspase recruitment domain and the p19 domains of the caspase-2 zymogen. However, the structural details of this interaction and the exact role of 14-3-3 in the regulation of caspase-2 activation remain unclear. Moreover, the caspase-2 region with both 14-3-3-binding motifs also contains the nuclear localization sequence (NLS), thus suggesting that 14-3-3 binding may regulate the subcellular localization of caspase-2. Here, we report a structural analysis of the 14-3-3ζ:caspase-2 complex using a combined approach based on small angle X-ray scattering, NMR, chemical cross-linking, and fluorescence spectroscopy. The structural model proposed in this study suggests that phosphorylated caspase-2 and 14-3-3ζ form a compact and rigid complex in which the p19 and the p12 domains of caspase-2 are positioned within the central channel of the 14-3-3 dimer and stabilized through interactions with the C-terminal helices of both 14-3-3ζ protomers. In this conformation, the surface of the p12 domain, which is involved in caspase-2 activation by dimerization, is sterically occluded by the 14-3-3 dimer, thereby likely preventing caspase-2 activation. In addition, 14-3-3 protein binding to caspase-2 masks its NLS. Therefore, our results suggest that 14-3-3 protein binding to caspase-2 may play a key role in regulating caspase-2 activation. DATABASE: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.ww pdb.org (PDB ID codes 6GKF and 6GKG).
- MeSH
- cysteinové endopeptidasy chemie metabolismus MeSH
- fosforylace MeSH
- jaderné lokalizační signály * MeSH
- kaspasa 2 chemie metabolismus MeSH
- konformace proteinů MeSH
- lidé MeSH
- maloúhlový rozptyl MeSH
- molekulární modely MeSH
- proteiny 14-3-3 chemie metabolismus MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
African trypanosomes are medically important parasites that cause sleeping sickness in humans and nagana in animals. In addition to their pathogenic role, they have emerged as valuable model organisms for studying fundamental biological processes. Protein tagging is a powerful tool for investigating protein localization and function. In a previous study, we developed two plasmids for rapid and reproducible polymerase chain reaction-based protein tagging in trypanosomes, which enabled the subcellular mapping of 89% of the trypanosome proteome. However, the limited selection of fluorescent protein tags and selectable markers restricted the flexibility of this approach. Here, we present an extended set of >100 plasmids that incorporate universal primer annealing sequences, enabling protein tagging with a range of fluorescent, biochemical and epitope tags, using five different selection markers. We evaluated the suitability of various fluorescent proteins for live and fixed cell imaging, fluorescent movies, and we demonstrate the use of tagging plasmids encoding tandem epitope tags to support expansion microscopy approaches. We show that this series of plasmids is functional in other trypanosomatid parasites, significantly increasing its value. Finally, we developed a new plasmid for tagging glycosylphosphatidylinositol-anchored proteins. We anticipate that this will be an important toolset for investigating trypanosomatid protein localization and function.
Studium protein‑proteinových interakcí in vivo se v současné době dostává do popředí zájmu – umožňuje prokázat nebo upřesnit již známé protein‑proteinové interakce a odhalit jejich inhibitory, zachytit konformační změny proteinů, objasnit nebo upřesnit signální kaskády v živé buňce s minimálním ovlivněním jejího buněčného prostředí. Jedním z možných přístupů umožňujících tuto charakteristiku jsou metody využívající rezonančního přenosu energie – fluorescenční (FRET) a jeho pozdější modifikace bioluminiscenční (BRET). Tyto metody jsou založeny na zviditelnění proteinových interakcí pomocí excitace fluorescenčních proteinů, ať už světelně nebo enzymaticky. Tyto přístupy umožňují nejen lokalizovat proteiny v buňce nebo jejich organelách (případně i v malých živočiších), ale i kvantifikovat intenzitu fluorescenčního nebo luminiscenčního signálu a odhalit pevnost vazby mezi interakčními partnery. V tomto příspěvku je objasněn princip metod FRET a BRET, jejich konkrétní aplikace při studiu protein‑proteinových interakcí a jsou popsány dosavadní poznatky získané s využitím těchto metod a upřesňující některé molekulární a buněčné mechanizmy a signalizace související s nádorovou biologií.
Nowadays, in vivo protein‑protein interaction studies have become preferable detecting methods that enable to show or specify (already known) protein interactions and discover their inhibitors. They also facilitate detection of protein conformational changes and discovery or specification of signaling pathways in living cells. One group of in vivo methods enabling these findings is based on fluorescent resonance energy transfer (FRET) and its bioluminescent modification (BRET). They are based on visualization of protein‑protein interactions via light or enzymatic excitation of fluorescent or bioluminescent proteins. These methods allow not only protein localization within the cell or its organelles (or small animals) but they also allow us to quantify fluorescent signals and to discover weak or strong interaction partners. In this review, we explain the principles of FRET and BRET, their applications in the characterization of protein‑protein interactions and we describe several findings using these two methods that clarify molecular and cellular mechanisms and signals related to cancer biology. Key words: FRET – BRET – imaging methods – protein‑protein interaction in vivo This work was supported by the Czech Science Foundation projects P206/12/G151 and 13--00956S, by the European Regional Development Fund and the State Budget of the Czech Republic (RECAMO, CZ.1.05/2.1.00/03.0101) and by MH CZ – DRO (MMCI, 00209805). The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE “uniform requirements” for biomedical papers. Submitted: 20. 1. 2014 Accepted: 31. 3. 2014
- Klíčová slova
- intramolekulární biosenzory, protein‑proteinové interakce in vivo, intermolekulární biosenzory,
- MeSH
- biosenzitivní techniky * MeSH
- fluorescence MeSH
- fluorescenční barviva MeSH
- mapování interakce mezi proteiny * metody MeSH
- rezonanční přenos fluorescenční energie * metody MeSH
- sbalování proteinů MeSH
- techniky přenosu energie bioluminiscenční rezonancí * metody MeSH
- zelené fluorescenční proteiny MeSH
- Publikační typ
- práce podpořená grantem MeSH
Trypanosoma brucei has a complex life cycle during which its single mitochondrion is subjected to major metabolic and morphological changes. While the procyclic stage (PS) of the insect vector contains a large and reticulated mitochondrion, its counterpart in the bloodstream stage (BS) parasitizing mammals is highly reduced and seems to be devoid of most functions. We show here that key Fe-S cluster assembly proteins are still present and active in this organelle and that produced clusters are incorporated into overexpressed enzymes. Importantly, the cysteine desulfurase Nfs, equipped with the nuclear localization signal, was detected in the nucleolus of both T. brucei life stages. The scaffold protein Isu, an interacting partner of Nfs, was also found to have a dual localization in the mitochondrion and the nucleolus, while frataxin and both ferredoxins are confined to the mitochondrion. Moreover, upon depletion of Isu, cytosolic tRNA thiolation dropped in the PS but not BS parasites.
- MeSH
- aktivní transport - buněčné jádro MeSH
- buněčné jádro metabolismus MeSH
- ferredoxiny metabolismus MeSH
- jaderné lokalizační signály MeSH
- lyasy štěpící vazby C-S chemie genetika metabolismus MeSH
- mitochondriální proteiny metabolismus MeSH
- mitochondrie metabolismus MeSH
- molekulární sekvence - údaje MeSH
- multimerizace proteinu MeSH
- proteiny asociované s jadernou matrix chemie genetika metabolismus MeSH
- proteiny vázající železo metabolismus MeSH
- protozoální proteiny chemie genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- Trypanosoma brucei brucei enzymologie genetika metabolismus MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The protein homologous to the tumor suppressor p53, p73, has essential roles in development and tumorigenesis. This protein exists in a wide range of isoforms with different, even antagonistic, functions. However, there are virtually no detailed morphological studies analyzing the endogenous expression of p73 isoforms at the cellular level in cancer cells. In this study, we investigated the expression and subcellular distribution of two N-terminal isoforms, TAp73 and ΔNp73, in medulloblastoma cells using immunofluorescence microscopy. Both proteins were observed in all cell lines examined, but differences were noted in their intracellular localization between the reference Daoy cell line and four newly established medulloblastoma cell lines (MBL-03, MBL-06, MBL-07 and MBL-10). In the new cell lines, TAp73 and ΔNp73 were located predominantly in cell nuclei. However, there was heterogeneity in TAp73 distribution in the cells of all MBL cell lines, with the protein located in the nucleus and also in a limited non-random area in the cytoplasm. In a small percentage of cells, we detected cytoplasmic localization of TAp73 only, i.e., nuclear exclusion was observed. Our results provide a basis for future studies on the causes and function of distinct intracellular localization of p73 protein isoforms with respect to different protein-protein interactions in medulloblastoma cells.
- MeSH
- buněčné jádro metabolismus MeSH
- dítě MeSH
- DNA vazebné proteiny chemie metabolismus MeSH
- intracelulární prostor metabolismus MeSH
- jaderné proteiny chemie metabolismus MeSH
- lidé MeSH
- meduloblastom metabolismus patologie MeSH
- nádorové buněčné linie MeSH
- nádorové supresorové proteiny chemie metabolismus MeSH
- předškolní dítě MeSH
- protein - isoformy metabolismus MeSH
- transport proteinů MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Mitogen-activated protein kinases (MAPK) are key regulatory elements in many processes. They are highly conserved throughout eukaryotes. In plants, MAPKs are involved in biotic and abiotic stress responses; they regulate cell division, cell growth, and also programmed cell death. In vivo visualization of MAPKs is crucial for understanding of their spatiotemporal organization. Cloning of MAPK-fluorescent protein fusions might present difficulties related to the preservation of protein-protein interactions essential for MAPK localization, interactions with upstream and downstream regulators, and finally substrate targeting. In this chapter we describe cloning of MAPKs in the flexible MultiSite Gateway(®) cloning system followed by easy and quick testing of binary vectors by transient assays in Arabidopsis thaliana and Nicotiana benthamiana.
- MeSH
- Agrobacterium genetika MeSH
- Arabidopsis enzymologie genetika MeSH
- cetrimoniové sloučeniny chemie MeSH
- DNA primery genetika MeSH
- DNA rostlinná genetika izolace a purifikace MeSH
- Escherichia coli genetika MeSH
- genetické inženýrství metody MeSH
- genetické vektory genetika MeSH
- genom rostlinný genetika MeSH
- klonování DNA MeSH
- listy rostlin genetika MeSH
- mitogenem aktivované proteinkinasy genetika metabolismus MeSH
- polymerázová řetězová reakce MeSH
- promotorové oblasti (genetika) genetika MeSH
- proteiny huseníčku genetika metabolismus MeSH
- rekombinantní fúzní proteiny genetika metabolismus MeSH
- tabák genetika MeSH
- transformace genetická MeSH
- transport proteinů MeSH
- zelené fluorescenční proteiny genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Protein p21(Cip1/Waf1) is a cyclin-dependent kinase inhibitor, which is important in the response of cells to genotoxic stress and a major transcriptional target of p53 protein. Based on the localization, p21(Cip1/Waf1) protein executes various functions in the cell. In the nucleus p21(Cip1/Waf1) binds to and inhibits the activity of cyclin dependent kinases Cdk1 and Cdk2 and blocks the transition from G1 phase into S phase or from G2 phase into mitosis after DNA damage. This enables the repair of damaged DNA. p21(Cip1/Waf1) was also found as an important protein for the induction of replication senescence as well as stress-induced premature senescence. In the cytoplasm, p21(Cip1/Waf1) protein has an anti-apoptotic effect. It is able to bind to and inhibit caspase 3, as well as the apoptotic kinases ASK1 and JNK. The function of p21(Cip1/Waf1) in response to a DNA damage probably depends on the extent of the damage. In the case of low-level DNA damage, the expression of p21(Cip1/Waf1) is increased, it induces cell cycle arrest, and performs also anti-apoptotic activities. However, after extensive DNA damage the amount of p21(Cip1/Waf1) protein is decreased and the cell undergoes apoptosis. Dual function of p21(Cip1/Waf1) was also observed in cancerogenesis. On the one hand, p21(Cip1/Waf1) acts as a tumor suppressor; on the other hand it prevents apoptosis and acts as an oncogene. Better understanding of the role of p21(Cip1/Waf1) in various conditions would help to develop better cancer-treatment strategies.
- MeSH
- buněčné jádro metabolismus MeSH
- cytoplazma metabolismus MeSH
- inhibitor p21 cyklin-dependentní kinasy metabolismus MeSH
- lidé MeSH
- poškození DNA MeSH
- posttranslační úpravy proteinů MeSH
- subcelulární frakce metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
