Tumor suppressor p53 is a key player in the cell response to DNA damage that suffers by frequent inactivating aberrations. Some of them disturb p53 oligomerization and influence cell decision between proliferation, growth arrest and apoptosis. Active p53 resides mostly in the nucleus, degradation occurs in the cytoplasm. Acute myeloid leukemia (AML)-related mutation of NPM (NPMmut) induces massive mislocalization of p53 to the cytoplasm, which might be related to leukemia initiation. Since both proteins interact and execute their function as oligomers, we investigated the role of perturbed p53 oligomerization in the p53 mislocalization process in live cells by FLIM (fluorescence lifetime imaging microscopy), fluorescence anisotropy imaging (FAIM), fluorescence cross-correlation spectroscopy (FCCS) and immunochemical methods. On a set of fluorescently labeled p53 variants, monomeric R337G and L344P, dimeric L344A, and multimeric D352G and A353S, we correlated their cellular localization, oligomerization and interaction with NPMmut. Interplay between nuclear export signal (NES) and nuclear localization signal (NLS) of p53 was investigated as well. While NLS was found critical for the nuclear p53 localization, NES plays less significant role. We observed cytoplasmic translocation only for multimeric A353S variant with sufficient stability and strong interaction with NPMmut. Less stable multimer D352G and L344A dimer were not translocated, monomeric p53 variants always resided in the nucleus independently of the presence of NPMmut and NES intactness. Oligomeric state of NPMmut is not required for p53 translocation, which happens also in the presence of the nonoligomerizing NPMmut variant. The prominent structural and functional role of the R337 residue is shown.
- MeSH
- Leukemia, Myeloid, Acute * genetics metabolism MeSH
- Cell Nucleus metabolism MeSH
- Cytoplasm metabolism MeSH
- Nuclear Localization Signals metabolism MeSH
- Nuclear Proteins * genetics metabolism MeSH
- Humans MeSH
- Protein Multimerization MeSH
- Mutation * MeSH
- Cell Line, Tumor MeSH
- Tumor Suppressor Protein p53 * metabolism genetics chemistry MeSH
- Nucleophosmin MeSH
- Nuclear Export Signals MeSH
- Protein Transport MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Protein synthesis (translation) consumes a substantial proportion of cellular resources, prompting specialized mechanisms to reduce translation under adverse conditions. Ribosome inactivation often involves ribosome-interacting proteins. In both bacteria and eukaryotes, various ribosome-interacting proteins facilitate ribosome dimerization or hibernation, and/or prevent ribosomal subunits from associating, enabling the organisms to adapt to stress. Despite extensive studies on bacteria and eukaryotes, understanding factor-mediated ribosome dimerization or anti-association in archaea remains elusive. Here, we present cryo-electron microscopy structures of an archaeal 30S dimer complexed with an archaeal ribosome dimerization factor (designated aRDF), from Pyrococcus furiosus, resolved at a resolution of 3.2 Å. The complex features two 30S subunits stabilized by aRDF homodimers in a unique head-to-body architecture, which differs from the disome architecture observed during hibernation in bacteria and eukaryotes. aRDF interacts directly with eS32 ribosomal protein, which is essential for subunit association. The binding mode of aRDF elucidates its anti-association properties, which prevent the assembly of archaeal 70S ribosomes.
- MeSH
- Archaeal Proteins * chemistry metabolism ultrastructure MeSH
- Dimerization MeSH
- Cryoelectron Microscopy * MeSH
- Ribosome Subunits, Small, Archaeal chemistry metabolism MeSH
- Models, Molecular MeSH
- Protein Multimerization MeSH
- Pyrococcus furiosus * metabolism MeSH
- Ribosomal Proteins * chemistry metabolism MeSH
- Ribosomes metabolism ultrastructure chemistry MeSH
- Protein Binding MeSH
- Publication type
- Journal Article MeSH
Activation of the Wnt/β-catenin pathway crucially depends on the polymerization of dishevelled 2 (DVL2) into biomolecular condensates. However, given the low affinity of known DVL2 self-interaction sites and its low cellular concentration, it is unclear how polymers can form. Here, we detect oligomeric DVL2 complexes at endogenous protein levels in human cell lines, using a biochemical ultracentrifugation assay. We identify a low-complexity region (LCR4) in the C-terminus whose deletion and fusion decreased and increased the complexes, respectively. Notably, LCR4-induced complexes correlated with the formation of microscopically visible multimeric condensates. Adjacent to LCR4, we mapped a conserved domain (CD2) promoting condensates only. Molecularly, LCR4 and CD2 mediated DVL2 self-interaction via aggregating residues and phenylalanine stickers, respectively. Point mutations inactivating these interaction sites impaired Wnt pathway activation by DVL2. Our study discovers DVL2 complexes with functional importance for Wnt/β-catenin signaling. Moreover, we provide evidence that DVL2 condensates form in two steps by pre-oligomerization via high-affinity interaction sites, such as LCR4, and subsequent condensation via low-affinity interaction sites, such as CD2.
- MeSH
- beta Catenin metabolism genetics MeSH
- HEK293 Cells MeSH
- Humans MeSH
- Protein Multimerization MeSH
- Dishevelled Proteins * metabolism genetics MeSH
- Wnt Signaling Pathway * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Truncating genetic variants of SORL1, encoding the endosome recycling receptor SORLA, have been accepted as causal of Alzheimer's disease (AD). However, most genetic variants observed in SORL1 are missense variants, for which it is complicated to determine the pathogenicity level because carriers come from pedigrees too small to be informative for penetrance estimations. Here, we describe three unrelated families in which the SORL1 coding missense variant rs772677709, that leads to a p.Y1816C substitution, segregates with Alzheimer's disease. Further, we investigate the effect of SORLA p.Y1816C on receptor maturation, cellular localization, and trafficking in cell-based assays. Under physiological circumstances, SORLA dimerizes within the endosome, allowing retromer-dependent trafficking from the endosome to the cell surface, where the luminal part is shed into the extracellular space (sSORLA). Our results showed that the p.Y1816C mutant impairs SORLA homodimerization in the endosome, leading to decreased trafficking to the cell surface and less sSORLA shedding. These trafficking defects of the mutant receptor can be rescued by the expression of the SORLA 3Fn-minireceptor. Finally, we find that iPSC-derived neurons with the engineered p.Y1816C mutation have enlarged endosomes, a defining cytopathology of AD. Our studies provide genetic as well as functional evidence that the SORL1 p.Y1816C variant is causal for AD. The partial penetrance of the mutation suggests this mutation should be considered in clinical genetic screening of multiplex early-onset AD families.
- MeSH
- Alzheimer Disease * genetics metabolism pathology MeSH
- Endosomes * metabolism MeSH
- HEK293 Cells MeSH
- Middle Aged MeSH
- Humans MeSH
- Membrane Transport Proteins * genetics metabolism MeSH
- Mutation, Missense MeSH
- Protein Multimerization MeSH
- LDL-Receptor Related Proteins * genetics metabolism MeSH
- Pedigree * MeSH
- Aged MeSH
- Protein Transport MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
Several peripheral membrane proteins are known to form membrane pores through multimerization. In many cases, in biochemical reconstitution experiments, a complex distribution of oligomeric states has been observed that may, in part, be irrelevant to their physiological functions. This phenomenon makes it difficult to identify the functional oligomeric states of membrane lipid interacting proteins, for example, during the formation of transient membrane pores. Using fibroblast growth factor 2 (FGF2) as an example, we present a methodology applicable to giant lipid vesicles by which functional oligomers can be distinguished from nonspecifically aggregated proteins without functionality. Two distinct populations of fibroblast growth factor 2 were identified with (i) dimers to hexamers and (ii) a broad population of higher oligomeric states of membrane-associated FGF2 oligomers significantly distorting the original unfiltered histogram of all detectable oligomeric species of FGF2. The presented statistical approach is relevant for various techniques for characterizing membrane-dependent protein oligomerization.
14-3-3 proteins are universal regulatory proteins and their function depends on their oligomeric form which may alter between the monomeric, homodimeric and heterodimeric states. The populations of individual oligomeric forms are controlled by Kd values of the dimer-monomer equilibria between the involved isoforms. This complex picture is extended by post-translational modifications, e.g. phosphorylation. In this work, we describe the equilibria between monomers, homo- and heterodimers of the 14-3-3ζ isoform in the unmodified and phosphorylated form. To cover a wide range of dimerization affinities, we combined solution NMR, microscale thermophoresis, native PAGE, and a set of novel fluorescence assays. Using a FRET based assay, we also determined the kinetic parameters of dimerization. We found that phosphorylation of 14-3-3ζ at Ser58 increases its homodimeric Kd value by 6 orders of magnitude. The presented assays allow to efficiently monitor 14-3-3ζ dimerization as a function of external factors, such as temperature, salt concentration, and client protein binding. For instance, we obtained values of both transient and equilibrium thermodynamic constants for the dimerization, and observed a substantial decrease of 14-3-3ζ dimer dissociation rate upon binding to the doubly phosphorylated regulatory domain of tyrosine hydroxylase. In summary, our work provides a conceptual framework to characterise the isoform exchanges of homo- and heterodimers which can significantly deepen our knowledge about the regulatory function of 14-3-3 proteins.
- MeSH
- Phosphorylation MeSH
- Humans MeSH
- Protein Multimerization MeSH
- 14-3-3 Proteins * chemistry MeSH
- Thermodynamics MeSH
- Protein Binding MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Life on Earth depends on photosynthesis, the conversion of light energy into chemical energy. Plants collect photons by light harvesting complexes (LHC)-abundant membrane proteins containing chlorophyll and xanthophyll molecules. LHC-like proteins are similar in their amino acid sequence to true LHC antennae, however, they rather serve a photoprotective function. Whether the LHC-like proteins bind pigments has remained unclear. Here, we characterize plant LHC-like proteins (LIL3 and ELIP2) produced in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). Both proteins were associated with chlorophyll a (Chl) and zeaxanthin and LIL3 was shown to be capable of quenching Chl fluorescence via direct energy transfer from the Chl Qy state to zeaxanthin S1 state. Interestingly, the ability of the ELIP2 protein to quench can be acquired by modifying its N-terminal sequence. By employing Synechocystis carotenoid mutants and site-directed mutagenesis we demonstrate that, although LIL3 does not need pigments for folding, pigments stabilize the LIL3 dimer.
- MeSH
- Chlorophyll metabolism MeSH
- Carotenoids metabolism MeSH
- Protein Multimerization MeSH
- Mutation MeSH
- Energy Transfer MeSH
- Chloroplast Proteins chemistry genetics metabolism MeSH
- Arabidopsis Proteins chemistry genetics metabolism MeSH
- Protein Folding MeSH
- Synechocystis genetics metabolism MeSH
- Protein Binding MeSH
- Xanthophylls metabolism MeSH
- Zeaxanthins genetics metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Opioid receptors (ORs) have been observed as homo- and heterodimers, but it is unclear if the dimers are stable under physiological conditions, and whether monomers or dimers comprise the predominant fraction in a cell. Here, we use three live-cell imaging approaches to assess dimerization of ORs at expression levels that are 10-100 × smaller than in classical biochemical assays. At membrane densities around 25/µm2, a split-GFP assay reveals that κOR dimerizes, while µOR and δOR stay monomeric. At receptor densities < 5/µm2, single-molecule imaging showed no κOR dimers, supporting the concept that dimer formation depends on receptor membrane density. To directly observe the transition from monomers to dimers, we used a single-molecule assay to assess membrane protein interactions at densities up to 100 × higher than conventional single-molecule imaging. We observe that κOR is monomeric at densities < 10/µm2 and forms dimers at densities that are considered physiological. In contrast, µOR and δOR stay monomeric even at the highest densities covered by our approach. The observation of long-lasting co-localization of red and green κOR spots suggests that it is a specific effect based on OR dimerization and not an artefact of coincidental encounters.
- MeSH
- Single-Cell Analysis methods MeSH
- Cell Membrane metabolism MeSH
- Protein Conformation MeSH
- Rats MeSH
- Protein Multimerization MeSH
- Mice MeSH
- Receptors, Opioid, delta chemistry metabolism MeSH
- Receptors, Opioid, mu chemistry metabolism MeSH
- Single Molecule Imaging methods MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Protein-protein interactions (PPIs) remain poorly explored targets for the treatment of Alzheimer's disease. The interaction of 14-3-3 proteins with Tau was shown to be linked to Tau pathology. This PPI is therefore seen as a potential target for Alzheimer's disease. When Tau is phosphorylated by PKA (Tau-PKA), several phosphorylation sites are generated, including two known 14-3-3 binding sites, surrounding the phosphorylated serines 214 and 324 of Tau. The crystal structures of 14-3-3 in complex with peptides surrounding these Tau phosphosites show that both these motifs are anchored in the amphipathic binding groove of 14-3-3. However, in the absence of structural data with the full-length Tau protein, the stoichiometry of the complex or the interface and affinity of the partners is still unclear. In this work, we addressed these points, using a broad range of biophysical techniques. The interaction of the long and disordered Tau-PKA protein with 14-3-3σ is restricted to two short sequences, containing phosphorylated serines, which bind in the amphipathic binding groove of 14-3-3σ. Phosphorylation of Tau is fundamental for the formation of this stable complex, and the affinity of the Tau-PKA/14-3-3σ interaction is in the 1-10 micromolar range. Each monomer of the 14-3-3σ dimer binds one of two different phosphorylated peptides of Tau-PKA, suggesting a 14-3-3/Tau-PKA stoichiometry of 2 : 1, confirmed by analytical ultracentrifugation. These results contribute to a better understanding of this PPI and provide useful insights for drug discovery projects aiming at the modulation of this interaction.
- MeSH
- Alzheimer Disease genetics metabolism pathology MeSH
- Exoribonucleases chemistry genetics metabolism MeSH
- Phosphorylation MeSH
- Protein Conformation MeSH
- Humans MeSH
- Magnetic Resonance Spectroscopy MeSH
- Protein Multimerization * MeSH
- Mutation MeSH
- Surface Plasmon Resonance MeSH
- Cyclic AMP-Dependent Protein Kinases metabolism MeSH
- 14-3-3 Proteins chemistry genetics metabolism MeSH
- tau Proteins chemistry genetics metabolism MeSH
- Serine chemistry metabolism MeSH
- Protein Binding MeSH
- Binding Sites MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Mounting evidence suggests that the neuronal cell membrane is the main site of oligomer-mediated neuronal toxicity of amyloid-β peptides in Alzheimer's disease. To gain a detailed understanding of the mutual interference of amyloid-β oligomers and the neuronal membrane, we carried out microseconds of all-atom molecular dynamics (MD) simulations on the dimerization of amyloid-β (Aβ)42 in the aqueous phase and in the presence of a lipid bilayer mimicking the in vivo composition of neuronal membranes. The dimerization in solution is characterized by a random coil to β-sheet transition that seems on pathway to amyloid aggregation, while the interactions with the neuronal membrane decrease the order of the Aβ42 dimer by attenuating its propensity to form a β-sheet structure. The main lipid interaction partners of Aβ42 are the surface-exposed sugar groups of the gangliosides GM1. As the neurotoxic activity of amyloid oligomers increases with oligomer order, these results suggest that GM1 is neuroprotective against Aβ-mediated toxicity.
- MeSH
- Amyloid chemistry MeSH
- Amyloid beta-Peptides chemistry metabolism MeSH
- Cell Membrane metabolism MeSH
- G(M1) Ganglioside metabolism MeSH
- Protein Conformation MeSH
- Humans MeSH
- Lipid Bilayers metabolism MeSH
- Protein Multimerization * MeSH
- Neurons metabolism MeSH
- Molecular Dynamics Simulation MeSH
- Protein Binding MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
