oligomerization Dotaz Zobrazit nápovědu
Mnoho buněčných proteinů tvoří oligomery. Rovnováha mezi monomerním a oligomerním stavem těchto proteinů hraje důležitou roli v regulaci proteinové aktivity. Ovlivnění oligomerizace se tudíž nabízí jako zajímavý přístup při vývoji nových terapeutických látek. Cílem tohoto přehledového článku je shrnout informace o procesu proteinové oligomerizace a možnostech její cílené modulace. Role oligomerizace v onkogenezi je prezentována na příkladu nádorového supresorového proteinu p53, u kterého jsou v současné době zkoumány látky stabilizující jeho tetramerní strukturu. Z metod pro studium oligomerizace je zde představena metoda vodík/deuteriové výměny ve spojení s hmotnostní spektrometrií, která je vhodná pro detekci protein‑proteinových interakcí a analýzu dynamiky oligomerizace.
Many cellular proteins form oligomers. The equilibrium between monomeric and oligomeric states of these proteins is important for the regulation of protein activity. Modulation of the oligomerization equilibrium could be an interesting approach in the development of new therapeutic agents. This review summarizes information about protein oligomerization and modulation of this process, demonstrating the role of oligomerization in oncogenesis by tumor suppressor protein p53, which forms tetrameric structure. Today, many studies focus on finding compounds that stabilize its tetramers. Among the methods for studying oligomerization, we present hydrogen/deuterium exchange method coupled with mass spectrometry which is suitable for the detection of protein‑protein interaction and analysis of oligomerization dynamics. Key words: proteomics – drug design – tumor suppressor protein p53 – oligomerization – hydrogen/deuterium exchange This study was supported by the European Regional Development Fund and the State Budget of the Czech Republic (RECAMO, CZ.1.05/2.1.00/03.0101), MEYS – NPS I – LO1413, MH CZ – DRO (MMCI, 00209805) and BBMRI_CZ (LM2010004). The authors declare they have no potential confl icts 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: 9. 4. 2015 Accepted: 15. 6. 2015
- Klíčová slova
- vodík/deuteriová výměna,
- MeSH
- deuterium MeSH
- konformace proteinů MeSH
- lidé MeSH
- nádorový supresorový protein p53 * MeSH
- objevování léků MeSH
- proteiny chemie MeSH
- proteomika * MeSH
- sbalování proteinů MeSH
- vodík-deuteriová výměna * MeSH
- vodík MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
Oligomerization plays a crucial role in the function of nucleophosmin (NPM), an abundant nucleolar phosphoprotein. Two dual-color methods based on modern fluorescence confocal microscopy are applied for tracking NPM aggregates in live cells: cross-correlation Number and Brightness analysis (ccN&B) combined with pulsed interleaved excitation (PIE) and fluorescence-lifetime imaging microscopy (FLIM) utilizing resonance energy transfer (FRET). HEK-293T cells were transfected with mixture of plasmids designed for tagging with fluorescent proteins so that the cells express mixed population of NPM labeled either with eGFP or mRFP1. We observe joint oligomers formed from the fluorescently labeled NPM. Having validated the in vivo methods, we study an effect of substitutions in cysteine 21 (Cys21) of the NPM N-terminus on the oligomerization to demonstrate applicability of the methods. Inhibitory effect of mutations of the Cys21 to nonpolar Ala or to aromatic Phe on the oligomerization was reported in literature using in vitro semi-native electrophoresis. However, we do not detect any break-up of the joint NPM oligomers due to the Cys21 mutations in live cells. In vivo microscopy observations are supported by an in vitro method, the GFP-Trap immunoprecipitation assay. Our results therefore show importance of utilizing several methods for detection of biologically relevant protein aggregates. In vivo monitoring of the NPM oligomerization, a potential cancer therapy target, by the presented methods offers a new way to monitor effects of drugs that are tested as NPM oligomerization inhibitors directly in live cells.
- MeSH
- jaderné proteiny metabolismus MeSH
- lidé MeSH
- proteinové agregáty fyziologie MeSH
- rezonanční přenos fluorescenční energie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Recent studies illustrate that short oligonucleotide sequences can be easily produced from nucleotide precursors in a template-free non-enzymatic way under dehydrating conditions, i.e. using essentially dry materials. Here we report that 3',5' cyclic AMP may also serve as a substrate of the reaction, which proceeds under moderate conditions yet with a lower efficiency than the previously reported oligomerization of 3',5' cyclic GMP. Optimally the oligomerization requires (i) a temperature of 80°C, (ii) a neutral to alkaline environment and (iii) a time on the order of weeks. Differences in the yield and required reaction conditions of the oligomerizations utilizing 3',5' cGMP and cAMP are discussed in terms of the crystal structures of the compounds. Polymerization of 3',5' cyclic nucleotides, whose paramount relevance in a prebiotic chemistry context has been widely accepted for decades, supports the possibility that the origin of extant genetic materials might have followed a direct uninterrupted path since its very beginning, starting from non-elaborately pre-activated monomer compounds and simple reactions.
Nucleophosmin (NPM) mutations causing its export from the nucleoli to the cytoplasm are frequent in acute myeloid leukemia (AML). Due to heterooligomerization of wild type NPM with the AML-related mutant, the wild-type becomes misplaced from the nucleoli and its functions are significantly altered. Dissociation of NPM heterooligomers may thus restore the proper localization and function of wild-type NPM. NSC348884 is supposed to act as a potent inhibitor of NPM oligomerization. The effect of NSC348884 on the NPM oligomerization was thoroughly examined by fluorescence lifetime imaging with utilization of FRET and by a set of immunoprecipitation and electrophoretic methods. Leukemia-derived cell lines and primary AML cells as well as cells transfected with fluorescently labeled NPM forms were investigated. Our results clearly demonstrate that NSC348884 does not inhibit formation of NPM oligomers neither in vivo nor in vitro. Instead, we document that NSC348884 cytotoxicity is rather associated with modified cell adhesion signaling. The cytotoxic mechanism of NSC348884 has therefore to be reconsidered.
- MeSH
- apoptóza účinky léků MeSH
- HEK293 buňky MeSH
- indoly farmakologie MeSH
- jaderné proteiny genetika metabolismus MeSH
- leukemie farmakoterapie genetika metabolismus MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- protinádorové látky farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
p53 is a tetrameric protein with a thermodynamically unstable deoxyribonucleic acid (DNA)-binding domain flanked by intrinsically disordered regulatory domains that control its activity. The unstable and disordered segments of p53 allow high flexibility as it interacts with binding partners and permits a rapid on/off switch to control its function. The p53 tetramer can exist in multiple conformational states, any of which can be stabilized by a particular modification. Here, we apply the allostery model to p53 to ask whether evidence can be found that the "activating" C-terminal phosphorylation of p53 stabilizes a specific conformation of the protein in the absence of DNA. We take advantage of monoclonal antibodies for p53 that measure indirectly the following conformations: unfolded, folded, and tetrameric. A double antibody capture enzyme linked-immunosorbent assay was used to observe evidence of conformational changes of human p53 upon phosphorylation by casein kinase 2 in vitro. It was demonstrated that oligomerization and stabilization of p53 wild-type conformation results in differential exposure of conformational epitopes PAb1620, PAb240, and DO12 that indicates a reduction in the "unfolded" conformation and increases in the folded conformation coincide with increases in its oligomerization state. These data highlight that the oligomeric conformation of p53 can be stabilized by an activating enzyme and further highlight the utility of the allostery model when applied to understanding the regulation of unstable and intrinsically disordered proteins.
- MeSH
- adenosintrifosfát metabolismus MeSH
- alosterická regulace MeSH
- fosforylace MeSH
- kaseinkinasa II metabolismus MeSH
- lidé MeSH
- molekulární modely MeSH
- multimerizace proteinu MeSH
- mutace MeSH
- nádorový supresorový protein p53 chemie genetika metabolismus MeSH
- proteinové domény MeSH
- stabilita proteinů MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The Bordetella adenylate cyclase-hemolysin (CyaA, ACT, or AC-Hly) is a multifunctional toxin. Simultaneously with promoting calcium ion entry, CyaA delivers into host cells an adenylate cyclase enzyme (AC) and permeabilizes cell membrane by forming small cation-selective pores. Indirect evidence suggested that these two activities were accomplished by different membrane-inserted CyaA conformers, one acting as an AC-delivering monomer and the other as an uncharacterized pore-forming oligomer. We tested this model by directly detecting toxin oligomers in cell membrane and by assessing oligomerization of specific mutants with altered pore-forming properties. CyaA oligomers were revealed in sheep erythrocyte membranes by immunogold labeling and directly demonstrated by pulldown of membrane-inserted CyaA together with biotinylated CyaA-AC(-) toxoid. Membrane oligomers of CyaA could also be resolved by nondenaturing electrophoresis of mild detergent extracts of erythrocytes. Furthermore, CyaA mutants exhibiting enhanced (E581K) or reduced (E570K+E581P) specific hemolytic and pore-forming activity were found to exhibit also a correspondingly enhanced or reduced propensity to form oligomers in erythrocyte membranes. On the other hand, processed CyaA, with the AC domain cleaved off by erythrocyte proteases, was detected only in a monomeric form excluded from the oligomers of unprocessed CyaA. These results provide the first direct evidence that oligomerization is involved in formation of CyaA pores in target membranes and that translocation of the AC domain across cell membrane may be accomplished by monomeric CyaA.
- MeSH
- adenylátcyklasový toxin farmakokinetika metabolismus MeSH
- Bordetella enzymologie MeSH
- endocytóza MeSH
- erytrocyty MeSH
- hemolýza účinky léků MeSH
- missense mutace MeSH
- multimerizace proteinu MeSH
- ovce MeSH
- permeabilita buněčné membrány účinky léků MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
NKp30 is one of the main human natural killer (NK) cell activating receptors used in directed immunotherapy. The oligomerization of the NKp30 ligand binding domain depends on the length of the C-terminal stalk region, but our structural knowledge of NKp30 oligomerization and its role in signal transduction remains limited. Moreover, ligand binding of NKp30 is affected by the presence and type of N-glycosylation. In this study, we assessed whether NKp30 oligomerization depends on its N-glycosylation. Our results show that NKp30 forms oligomers when expressed in HEK293S GnTI- cell lines with simple N-glycans. However, NKp30 was detected only as monomers after enzymatic deglycosylation. Furthermore, we characterized the interaction between NKp30 and its best-studied cognate ligand, B7-H6, with respect to glycosylation and oligomerization, and we solved the crystal structure of this complex with glycosylated NKp30, revealing a new glycosylation-induced mode of NKp30 dimerization. Overall, this study provides new insights into the structural basis of NKp30 oligomerization and explains how the stalk region and glycosylation of NKp30 affect its ligand affinity. This furthers our understanding of the molecular mechanisms involved in NK cell activation, which is crucial for the successful design of novel NK cell-based targeted immunotherapeutics.
- Publikační typ
- časopisecké články MeSH
