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.

• Klíčová slova
• CK2, allosteric regulation, conformational change, oligomerization, p53, phosphorylation, protein conformation, protein folding,
• 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
• Názvy látek
• adenosintrifosfát MeSH
• kaseinkinasa II MeSH
• nádorový supresorový protein p53 MeSH
• TP53 protein, human MeSH Prohlížeč

In-membrane oligomerization is decisive for the function (or dysfunction) of many proteins. Techniques were developed to characterize membrane-inserted oligomers and the hereby obtained oligomerization states were intuitively related to the function of these proteins. However, in many cases, it is unclear whether the obtained oligomerization states are functionally relevant or are merely the consequence of nonspecific aggregation. Using fibroblast growth factor 2 (FGF2) as a model system, we addressed this methodological challenge. FGF2 oligomerizes in a PI(4,5)P2-dependent manner at the inner plasma membrane leaflet. This process results in membrane insertion and the formation of a lipidic membrane pore, the key intermediate in unconventional secretion of FGF2. To tackle the problem of discriminating functional oligomers from irrelevant aggregates, we present a statistical single molecule and single vesicle assay determining the brightness of individually diffusing in-membrane oligomers and correlating their oligomerization state with membrane pore formation. Importantly, time-dependent membrane pore formation was analyzed with an ensemble of single vesicles providing detailed statistics. Our findings demonstrate that quantifying oligomeric states alone does not allow for a deep understanding of the structure-function relationship of membrane-inserted oligomers.

• MeSH
• buněčná membrána chemie   metabolismus   MeSH
• fibroblastový růstový faktor 2 chemie   metabolismus   MeSH
• fluorescenční spektrometrie MeSH
• kvarterní struktura proteinů MeSH
• multimerizace proteinu * MeSH
• permeabilita MeSH
• poréznost MeSH
• unilamelární lipozómy chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fibroblastový růstový faktor 2 MeSH
• unilamelární lipozómy MeSH

The oligomerization capacity of the retroviral matrix protein is an important feature that affects assembly of immature virions and their interaction with cellular membrane. A combination of NMR relaxation measurements and advanced analysis of molecular dynamics simulation trajectory provided an unprecedentedly detailed insight into internal mobility of matrix proteins of the Mason-Pfizer monkey virus. Strong evidence have been obtained that the oligomerization capacity of the wild-type matrix protein is closely related to the enhanced dynamics of several parts of its backbone on a nanosecond time scale. Increased flexibility has been observed for two regions: the loop between α-helices α2 and α3 and the C-terminal half of α-helix α3 which accommodate amino acid residues that form the oligomerization interface. On the other hand, matrix mutant R55F that has changed structure and does not exhibit any specific oligomerization in solution was found considerably more rigid. Our results document that conformational selection mechanism together with induced fit and favorable structural preorganization play an important role in the control of the oligomerization process.

• MeSH
• aminokyseliny chemie   genetika   MeSH
• kvarterní struktura proteinů MeSH
• Masonův-Pfizerův opičí virus chemie   MeSH
• multimerizace proteinu * MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• proteiny virové matrix chemie   MeSH
• simulace molekulární dynamiky MeSH
• substituce aminokyselin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• aminokyseliny MeSH
• proteiny virové matrix 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
• nukleofosmin MeSH
• proteinové agregáty fyziologie   MeSH
• rezonanční přenos fluorescenční energie metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• jaderné proteiny MeSH
• NPM1 protein, human MeSH Prohlížeč
• nukleofosmin MeSH
• proteinové agregáty MeSH

Reptin is a member of the AAA+ superfamily whose members can exist in equilibrium between monomeric apo forms and ligand bound hexamers. Inter-subunit protein-protein interfaces that stabilize Reptin in its oligomeric state are not well-defined. A self-peptide binding assay identified a protein-peptide interface mapping to an inter-subunit "rim" of the hexamer bridged by Tyrosine-340. A Y340A mutation reduced ADP-dependent oligomer formation using a gel filtration assay, suggesting that Y340 forms a dominant oligomer stabilizing side chain. The monomeric ReptinY340A mutant protein exhibited increased activity to its partner protein AGR2 in an ELISA assay, further suggesting that hexamer formation can preclude certain protein interactions. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) demonstrated that the Y340A mutation attenuated deuterium suppression of Reptin in this motif in the presence of ligand. By contrast, the tyrosine motif of Reptin interacts with a shallower pocket in the hetero-oligomeric structure containing Pontin and HDX-MS revealed no obvious role of the Y340 side chain in stabilizing the Reptin-Pontin oligomer. Molecular dynamic simulations (MDS) rationalized how the Y340A mutation impacts upon a normally stabilizing inter-subunit amino acid contact. MDS also revealed how the D299N mutation can, by contrast, remove oligomer de-stabilizing contacts. These data suggest that the Reptin interactome can be regulated by a ligand dependent equilibrium between monomeric and hexameric forms through a hydrophobic inter-subunit protein-protein interaction motif bridged by Tyrosine-340. SIGNIFICANCE: Discovering dynamic protein-protein interactions is a fundamental aim of research in the life sciences. An emerging view of protein-protein interactions in higher eukaryotes is that they are driven by small linear polypeptide sequences; the linear motif. We report on the use of linear-peptide motif screens to discover a relatively high affinity peptide-protein interaction for the AAA+ and pro-oncogenic protein Reptin. This peptide interaction site was shown to form a 'hot-spot' protein-protein interaction site, and validated to be important for ligand-induced oligomerization of the Reptin protein. These biochemical data provide a foundation to understand how single point mutations in Reptin can impact on its oligomerization and protein-protein interaction landscape.

• MeSH
• AAA doména * MeSH
• ATPázy spojené s různými buněčnými aktivitami chemie   metabolismus   MeSH
• DNA-helikasy chemie   metabolismus   MeSH
• interakční proteinové domény a motivy fyziologie   MeSH
• lidé MeSH
• molekulární chaperony chemie   metabolismus   MeSH
• multimerizace proteinu * MeSH
• mutace MeSH
• simulace molekulární dynamiky MeSH
• transportní proteiny chemie   metabolismus   MeSH
• tyrosin genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ATPázy spojené s různými buněčnými aktivitami MeSH
• DNA-helikasy MeSH
• molekulární chaperony MeSH
• RUVBL2 protein, human MeSH Prohlížeč
• transportní proteiny MeSH
• tyrosin MeSH

For most retroviruses, including HIV, association with the plasma membrane (PM) promotes the assembly of immature particles, which occurs simultaneously with budding and maturation. In these viruses, maturation is initiated by oligomerization of polyprotein precursors. In contrast, several retroviruses, such as Mason-Pfizer monkey virus (M-PMV), assemble in the cytoplasm into immature particles that are transported across the PM. Therefore, protease activation and specific cleavage must not occur until the pre-assembled particle interacts with the PM. This interaction is triggered by a bipartite signal consisting of a cluster of basic residues in the matrix (MA) domain of Gag polyprotein and a myristoyl moiety N-terminally attached to MA. Here, we provide evidence that myristoyl exposure from the MA core and its insertion into the PM occurs in M-PMV. By a combination of experimental methods, we show that this results in a structural change at the C-terminus of MA allowing efficient cleavage of MA from the downstream region of Gag. This suggests that, in addition to the known effect of the myristoyl switch of HIV-1 MA on the multimerization state of Gag and particle assembly, the myristoyl switch may have a regulatory role in initiating sequential cleavage of M-PMV Gag in immature particles.

• Klíčová slova
• betaretrovirus, infectious disease, matrix protein, maturation, microbiology, myristoyl switch, protease, viruses,
• MeSH
• buněčná membrána MeSH
• endopeptidasy MeSH
• genové produkty gag chemie   MeSH
• Masonův-Pfizerův opičí virus * chemie   fyziologie   MeSH
• proteiny MeSH
• sestavení viru MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• endopeptidasy MeSH
• genové produkty gag MeSH
• proteiny MeSH

β-Amyloid (Aβ) oligomers are neurotoxic and implicated in Alzheimer's disease. Neuronal plasma membranes may mediate formation of Aβ oligomers in vivo. Membrane components sphingomyelin and GM1 have been shown to promote aggregation of Aβ; however, these studies were performed under extreme, non-physiological conditions. We demonstrate that physiological levels of GM1 , organized in nanodomains do not seed oligomerization of Aβ40 monomers. We show that sphingomyelin triggers oligomerization of Aβ40 and that GM1 is counteractive thus preventing oligomerization. We propose a molecular explanation that is supported by all-atom molecular dynamics simulations. The preventive role of GM1 in the oligomerization of Aβ40 suggests that decreasing levels of GM1 in the brain, for example, due to aging, could reduce protection against Aβ oligomerization and contribute to the onset of Alzheimer's disease.

• Klíčová slova
• Alzheimer's disease, amyloid beta-peptides, diffusion coefficients, fluorescence spectroscopy, neuroprotectives,
• MeSH
• amyloidní beta-protein antagonisté a inhibitory   metabolismus   MeSH
• G(M1) gangliosid chemie   farmakologie   MeSH
• sfingomyeliny chemie   farmakologie   MeSH
• simulace molekulární dynamiky MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• amyloidní beta-protein MeSH
• G(M1) gangliosid MeSH
• sfingomyeliny MeSH

BACKGROUND: Cartilage oligomeric matrix protein/thrombospondin 5 (COMP/TSP 5) is one of the most promising serologic markers with regard to an ability to prognose development of osteoarthritis (OA). Our aim was to map the epitopes of three monoclonal antibodies (mAb) to COMP and to develop and characterize a sandwich enzyme-linked immunosorbent assay (ELISA) for measuring COMP levels in human body fluids. METHODS: COMP was digested with trypsin and the NH(2)-terminal sequence of the fragments recognized by each of the mAbs was determined. Steric competition among the mAbs was tested with an antibody capture assay. A sandwich ELISA was developed using unlabeled mAb 16-F12 as a capture antibody, and mAb 17-C10 labeled with biotin as the second antibody. RESULTS: Epitopes of the three mAbs were mapped to three different domains within the COMP subunit (16-F12, NH(2)-terminal domain; 17-C10, EGF-like domain; 12-C4, COOH-terminal domain). These epitopes did not overlap. mAbs 17-C10 and 12-C4 yielded similar serum COMP results when used as the secondary antibodies. Serum COMP levels measured with the new sandwich ELISA using mAbs 16-F12 and 17-C10 correlated strongly with results based on an inhibition ELISA with mAb 17-C10 alone (r(2) = 0.836; P < 0.0001). We characterized the new sandwich ELISA with regards to inter- and intra-assay variability, the range of COMP levels that can be expected in human synovial fluids (SF) and sera (controls and OA and rheumatoid arthritis (RA) patients), and the day-to-day and diurnal variability of COMP levels in sera. CONCLUSIONS: We have developed and characterized a sandwich ELISA for COMP that is sensitive and yields highly reproducible COMP results upon analysis of human sera and synovial fluids.

• MeSH
• chrupavkový oligomerní matrixový protein MeSH
• ELISA metody   MeSH
• extracelulární matrix - proteiny analýza   imunologie   MeSH
• glykoproteiny analýza   imunologie   MeSH
• lidé MeSH
• mapování epitopu MeSH
• matriliny MeSH
• monoklonální protilátky imunologie   MeSH
• synoviální tekutina chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• chrupavkový oligomerní matrixový protein MeSH
• extracelulární matrix - proteiny MeSH
• glykoproteiny MeSH
• matriliny MeSH
• monoklonální protilátky MeSH
• TSP5 protein, human MeSH Prohlížeč

Human histone deacetylase 6 (HDAC6) is a structurally unique, multidomain protein implicated in a variety of physiological processes including cytoskeletal remodelling and the maintenance of cellular homeostasis. Our current understanding of the HDAC6 structure is limited to isolated domains, and a holistic picture of the full-length protein structure, including possible domain interactions, is missing. Here, we used an integrative structural biology approach to build a solution model of HDAC6 by combining experimental data from several orthogonal biophysical techniques complemented by molecular modelling. We show that HDAC6 is best described as a mosaic of folded and intrinsically disordered domains that in-solution adopts an ensemble of conformations without any stable interactions between structured domains. Furthermore, HDAC6 forms dimers/higher oligomers in a concentration-dependent manner, and its oligomerization is mediated via the positively charged N-terminal microtubule-binding domain. Our findings provide the first insights into the structure of full-length human HDAC6 and can be used as a basis for further research into structure function and physiological studies of this unique deacetylase.

• Klíčová slova
• acetylation, analytical ultracentrifugation, intrinsically disordered regions, oligomerization, small-angle X-ray scattering,
• MeSH
• acetylace MeSH
• histondeacetylasa 6 genetika   chemie   metabolismus   MeSH
• histondeacetylasy * metabolismus   MeSH
• inhibitory histondeacetylas MeSH
• lidé MeSH
• mikrotubuly * metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• histondeacetylasa 6 MeSH
• histondeacetylasy * MeSH
• inhibitory histondeacetylas MeSH

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
• nukleofosmin MeSH
• protinádorové látky farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• indoly MeSH
• jaderné proteiny MeSH
• NPM1 protein, human MeSH Prohlížeč
• NSC 348884 MeSH Prohlížeč
• nukleofosmin MeSH
• protinádorové látky MeSH