Formation Of Large Oligomers Research Articles

Contactin 2 homophilic adhesion structure and conformational plasticity

The cell-surface attached glycoprotein contactin 2 is ubiquitously expressed in the nervous system and mediates homotypic cell-cell interactions to organize cell guidance, differentiation, and adhesion. Contactin 2 consists of six Ig and four fibronectin type III domains (FnIII) of which the first four Ig domains form a horseshoe structure important for homodimerization and oligomerization. Here we report the crystal structure of the six-domain contactin 2Ig1-6 and show that the Ig5-Ig6 combination is oriented away from the horseshoe with flexion in interdomain connections. Two distinct dimer states, through Ig1-Ig2 and Ig3-Ig6 interactions, together allow formation of larger oligomers. Combined size exclusion chromatography with multiangle light scattering (SEC-MALS), small-angle X-ray scattering (SAXS) and native MS analysis indicates contactin 2Ig1-6 oligomerizes in a glycan dependent manner. SAXS and negative-stain electron microscopy reveals inherent plasticity of the contactin 2 full-ectodomain. The combination of intermolecular binding sites and ectodomain plasticity explains how contactin 2 can function as a homotypic adhesion molecule in diverse intercellular environments.

α-synuclein-assisted oligomerization of β-amyloid (1–42)

Alzheimer's disease (AD) and Parkinson's disease (PD) are the two most common neurodegenerative disorders, characterized by aggregation of amyloid polypeptides, β-amyloid (Aβ) and α-synuclein (αS), respectively. Aβ and αS follow similar aggregation pathways, starting from monomers, to soluble toxic oligomeric assemblies, and to insoluble fibrils. Various studies have suggested overlaps in the pathologies of AD and PD, and have shown Aβ-αS interactions. Unfortunately, whether these protein-protein interactions lead to self- and co-assembly of Aβ and αS into oligomers – a potentially toxic synergistic mechanism – is poorly understood. Among the various Aβ isoforms, interactions of Aβ containing 42 amino acids (Aβ (1–42), referred to as Aβ42) with αS are of most direct relevance due to the high aggregation propensity and the strong toxic effect of this Aβ isoform. In this study, we carefully determined molecular consequences of interactions between Aβ42 and αS in their respective monomeric, oligomeric, and fibrillar forms using a comprehensive set of experimental tools. We show that the three αS conformers, namely, monomers, oligomers and fibrils interfered with fibrillization of Aβ42. Specifically, αS monomers and oligomers promoted oligomerization and stabilization of soluble Aβ42, possibly via direct binding or co-assembly, while αS fibrils hindered soluble Aβ42 species from converting into insoluble aggregates by the formation of large oligomers. We also provide evidence that the interactions with αS were mediated by various parts of Aβ42, depending on Aβ42 and αS conformers. Furthermore, we compared similarities and dissimilarities between Aβ42-αS and Aβ40-αS interactions. Overall, the present study provides a comprehensive depiction of the molecular interplay between Aβ42 and αS, providing insight into its synergistic toxic mechanism.

The Role of the Arginine in the Conserved N-Terminal Domain RLFDQxFG Motif of Human Small Heat Shock Proteins HspB1, HspB4, HspB5, HspB6, and HspB8.

Although the N-terminal domain of vertebrate small heat shock proteins (sHsp) is poorly conserved, it contains a core motif preserved in many members of the sHsp family. The role of this RLFDQxFG motif remains elusive. We analyzed the specific role of the first arginine residue of this conserved octet sequence in five human sHsps (HspB1, HspB4, HspB5, HspB6, and HspB8). Substitution of this arginine with an alanine induced changes in thermal stability and/or intrinsic fluorescence of the related HspB1 and HspB8, but yielded only modest changes in the same biophysical properties of HspB4, HspB5, and HspB6 which together belong to another clade of vertebrate sHsps. Removal of the positively charged Arg side chain resulted in destabilization of the large oligomers of HspB1 and formation of smaller size oligomers of HspB5. The mutation induced only minor changes in the structure of HspB4 and HspB6. In contrast, the mutation in HspB8 was accompanied by shifting the equilibrium from dimers towards the formation of larger oligomers. We conclude that the RLFDQxFG motif plays distinct roles in the structure of several sHsp orthologs. This role correlates with the evolutionary relationship of the respective sHsps, but ultimately, it reflects the sequence context of this motif.

Structural Characterization of Human Pulmonary Surfactant Protein SP-D by Atomic Force Microscopy

Pulmonary surfactant protein D (SP-D), a C-type (Ca2+-dependent) lectin, belongs to the collectin family. It is found mainly in lung surfactant, participating in the innate immune defense of the lungs.SP-D monomer contains four structural domains: an N-terminus domain, a collagen region, an α-helical coiled-coil neck and a C-terminus carbohydrate recognition domain (CRD). Monomers form trimers through folding of the collagenous region into triple helices and the assembly of a coiled-coil bundle of α-helices in the neck region. These trimers are stabilized by two disulfide bonds in the cysteine-rich N-terminal domain. Trimers associate into higher order oligomers whose size and conformation is sensitive to environmental factors and the conditions during purification and storage. Despite extensive studies carried out to characterize the oligomerization process of SP-D, the pathway and type of interactions involved in the formation of large oligomers, are not clearly understood. In the current study, a structural characterization of recombinant human SP-D (rhSP-D), produced in mammalian CHO cells, has been carried out by Atomic Force Microscopy (AFM) and electrophoresis. The goal has been the determination of the distribution of oligomeric forms, the exploration of the possible oligomerization pathway and the description of the conformational diversity of rhSP-D. AFM experiments revealed that rhSP-D is a mixture of trimers, hexamers, dodecamers, and larger oligomeric species identified as “fuzzy balls”, with the most abundant structure being the dodecamer under the conditions of these experiments. We have developed a cross-linking protocol to detect the presence of SP-D dodecameric forms by PAGE-SDS, in which dodecamer is only visualized after chemical crosslinking and in the presence of denaturing agents, indicating the importance of hydrophobic interactions in dodecamer formation.

Identifying Oligomers and Lipid Vesicles Effects during α-Synuclein Fibril Formation through a Solid-State Nanopore

α-Synuclein (α-Syn) is a small intrinsically disordered 140 amino acids (14 kDa) protein expressed abundantly in human brain, regulating the release of neurotransmitters, vesicle recycling, and synaptic integrity. Adequate α-Syn monomers in solution undergo a nucleation-dependent mechanism growing into oligomers, which are able to elongate to form profibers through monomers addition and finally mature fibers. It is well acknowledged that the abnormal aggregation process of α-Syn monomers is associated with pathophysiology of Parkinson's disease. Identifying different oligomer types and tracking their aggregation dynamic process in lipid free or lipid co-incubated system, which not only present the significant step during fibrillation but also play a key role in pathogenesis of Parkinson's disease, is hard to know due to the inherent challenges of studying heterogeneous and transient system by using traditional biotechnology. In this study, we use solid-state nanopores to study the aggregation progress of α-Syn only sample and lipid-small unilamellar vesicles co-incubated α-Syn sample in a single-molecule approach. A tween 20 surface coating method was developed to enable continuous and smooth α-Syn translocation through SiN nanopores. Through analysis of translocation events for α-Syn incubated at different time, we identified four types of oligomers forming during aggregation process. These four types oligomers inferred as intermediates show a time-dependent quantity fraction change, suggesting that the formation of large oligomers is based on the consumption of small oligomers. Furthermore, we investigated the effect of lipid small unilamellar vesicles on α-Syn aggregation process. The experimental results indicate that it is the presence of 20% 1,2-dioleoyl-sn- glycero-3- [phospho-L-serine] (DOPS) dramatically enhances the aggregation rate of α-Syn, while the aggregation pathway is similar to α-Syn only samples.

Interaction of βA3-Crystallin with Deamidated Mutants of αA- and αB-Crystallins.

Interaction among crystallins is required for the maintenance of lens transparency. Deamidation is one of the most common post-translational modifications in crystallins, which results in incorrect interaction and leads to aggregate formation. Various studies have established interaction among the α- and β-crystallins. Here, we investigated the effects of the deamidation of αA- and αB-crystallins on their interaction with βA3-crystallin using surface plasmon resonance (SPR) and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) methods. SPR analysis confirmed adherence of WT αA- and WT αB-crystallins and their deamidated mutants with βA3-crystallin. The deamidated mutants of αA–crystallin (αA N101D and αA N123D) displayed lower adherence propensity for βA3-crystallin relative to the binding affinity shown by WT αA-crystallin. Among αB-crystallin mutants, αB N78D displayed higher adherence propensity whereas αB N146D mutant showed slightly lower binding affinity for βA3-crystallin relative to that shown by WT αB-crystallin. Under the in vivo condition (FLIM-FRET), both αA-deamidated mutants (αA N101D and αA N123D) exhibited strong interaction with βA3-crystallin (32±4% and 36±4% FRET efficiencies, respectively) compared to WT αA-crystallin (18±4%). Similarly, the αB N78D and αB N146D mutants showed strong interaction (36±4% and 22±4% FRET efficiencies, respectively) with βA3-crystallin compared to 18±4% FRET efficiency of WT αB-crystallin. Further, FLIM-FRET analysis of the C-terminal domain (CTE), N-terminal domain (NTD), and core domain (CD) of αA- and αB-crystallins with βA3-crystallin suggested that interaction sites most likely reside in the αA CTE and αB NTD regions, respectively, as these domains showed the highest FRET efficiencies. Overall, results suggest that similar to WT αA- and WTαB-crystallins, the deamidated mutants showed strong interactionfor βA3-crystallin. Variable in vitro and in vivo interactions are most likely due to the mutant’s large size oligomers, reduced hydrophobicity, and altered structures. Together, the results suggest that deamidation of α-crystallin may facilitate greater interaction and the formation of large oligomers with other crystallins, and this may contribute to the cataractogenic mechanism.

Cell Division Regulators MinC and MinD Form Polymers in the Presence of Nucleotide

The Min system of proteins, comprised of MinC, MinD and MinE, is essential for normal cell division in Escherichia coli. MinC forms an oscillating intracellular gradient near the poles that restricts placement of the division septum at midcell by inhibiting polymerization of FtsZ, the major component of the septal ring. MinC localization is directed by MinD, a Par-like ATPase that binds to the membrane in the presence of ATP. MinE functions as a regulator of MinD by stimulating MinD ATP hydrolysis, which leads to membrane dissociation. Results using purified proteins in vitro show that MinC stabilizes a conformational change in MinD in the presence of ATP characterized by the formation of large oligomers, which are detectable in light scattering and ultracentrifugation assays. Electron microscopy of MinC-MinD complexes formed with ATP show long polymers (300-500 nm) that are approximately 10-20 nm wide. Increasing the concentration of MinC in polymerization assays with MinD and ATP enhances oligomer formation. MinCD complexes assemble in the presence of the ATP analog ATPγS, but not ADP, suggesting that MinD is likely in an ATP-bound conformation. Using site-directed mutagenesis, we constructed a MinD mutant protein defective for ATP-dependent dimerization and a truncated MinD mutant protein lacking the membrane targeting sequence (MTS); both MinD mutant proteins are unable to polymerize with MinC. Additionally, the in vitro activity of MinC to prevent GTP-dependent FtsZ pelleting in sedimentation assays is impaired by MinD and ATP, suggesting polymerization with MinD may modulate MinC function. Finally, addition of MinE to MinC-MinD polymers induces concentration-dependent disassembly manner suggesting MinE competes with MinC for binding to MinD. Results suggest a novel nucleotide-dependent conformation of MinD with MinC and provides mechanistic insight into the functional interactions of the Min proteins.

Attempt to optimize some properties of fluorescent chimeras of human small heat shock protein HspB1 by modifying linker length and nature.

Chimerical proteins consisting of enhanced yellow fluorescent protein (EYFP) connected by linkers of different length and nature to the N-terminal end of small heat shock protein HspB1 were obtained and characterized. To obtain fluorescent chimeras with properties similar to those of unmodified small heat shock protein, we used either 12-residue-long linkers of different nature (highly flexible Gly-Ser linker (L1), rigid α-helical linker (L2), or rigid Pro-Ala linker (L3)) or highly flexible Gly-Ser linker consisting of 12, 18, or 21 residues. The wild-type HspB1 formed large stable oligomers consisting of more than 20 subunits. Independent of the length or the nature of the linker, all the fluorescent chimeras formed small (5-9 subunits) oligomers tending to dissociate at low protein concentration. Chaperone-like activity of the wild-type HspB1 and its fluorescent chimeras were compared using lysozyme as a model protein substrate. Under the conditions used, all the fluorescent chimeras possessed higher chaperone-like activity than the wild-type HspB1. Chaperone-like activity of fluorescent chimeras with L1 and L3 linkers was less different from that of the wild-type HspB1 compare to the chaperone-like activity of chimeras with rigid L2 linker. Increase in the length of L1 linker from 12 up to 21 residues leads to decrease in the difference in the chaperone-like activity between the wild-type protein and its fluorescent chimeras. Since the N-terminal domain of small heat shock proteins participates in formation of large oligomers, any way of attachment of fluorescent protein to the N-terminal end of HspB1 leads to dramatic changes in its oligomeric structure. Long flexible linkers should be used to obtain fluorescent chimeras with chaperone-like properties similar to those of the wild-type HspB1.

Bovine seminal ribonuclease triggers Beclin1-mediated autophagic cell death in pancreatic cancer cells

Among the large number of variants belonging to the pancreatic-type secretory ribonuclease (RNase) superfamily, bovine pancreatic ribonuclease (RNase A) is the proto-type and bovine seminal RNase (BS-RNase) represents the unique natively dimeric member. In the present manuscript, we evaluate the anti-tumoral property of these RNases in pancreatic adenocarcinoma cell lines and in nontumorigenic cells as normal control. We demonstrate that BS-RNase stimulates a strong anti-proliferative and pro-apoptotic effect in cancer cells, while RNase A is largely ineffective. Notably, we reveal for the first time that BS-RNase triggers Beclin1-mediated autophagic cancer cell death, providing evidences that high proliferation rate of cancer cells may render them more susceptible to autophagy by BS-RNase treatment. Notably, to improve the autophagic response of cancer cells to BS-RNase we used two different strategies: the more basic (as compared to WT enzyme) G38K mutant of BS-RNase, known to interact more strongly than wt with the acidic membrane of cancer cells, or BS-RNase oligomerization (tetramerization or formation of larger oligomers). Both mutant BS-RNase and BS-RNase oligomers potentiated autophagic cell death as compared to WT native dimer of BS-RNase, while the various RNase A oligomers remained completely ineffective. Altogether, our results shed more light on the mechanisms lying at the basis of BS-RNase antiproliferative effect in cancer cells, and support its potential use to develop new anti-cancer strategies.

New Physiological Function of Chaperones, Facilitating Reconstitution of Apoenzymes

One of the important branches of modern biochemistry and molecular biology is the investigation of structure and function of molecular chaperones. The main function of the family of molecular chaperones named as small heat shock proteins (sHsps) is suppression of aggregation of non-native protein species formed under stress conditions or during folding of the newly synthesized polypeptide chains [1-3]. The low molecular mass of monomers (from 12 to 43 kDa) and tendency to form large oligomers with high molecular masses up to 1000 kDa are typical of this protein family [4-14]. The presence of a conservative α-crystallin domain in the structure of sHsp seems to be important for the formation of stable dimers, whereas variable N- and C-terminal ends seem to participate in the formation of large oligomers [2-4,7]. It is supposed that the polydispersity and quaternary structure dynamics play an important role in cellular sHsp chaperones function [2,12]. sHsps cannot provide folding of the polypeptide chain; however, they form complexes with non-native proteins and can transfer the latter either to ATP-dependent chaperones that provide assistance to protein folding or proteasomes, where proteolytic degradation of the unfolded proteins occurs [15-19]

Pan‐BH3 Mimetic S1 Exhibits Broad‐Spectrum Antitumour Effects by Cooperation between Bax and Bak

Small molecule S1 is a pan-BH3 mimetic that can bind antiapoptotic Bcl-2, Bcl-xL and Mcl-1 proteins. Herein, different Bcl-2 member expression cancer cell lines (NCI-H345, MCF-7, SMMC-7721 and Hela) and cells deficient in Bax and/or Bak by shRNA were used to unravel the cascade of events by which S1 promotes apoptosis compared with Bcl-2/Bcl-xL inhibitor ABT-737. We identified that S1 exhibited broader antitumour spectrum than ABT-737 through disruption of more Bcl-2 interactions including Mcl-1/Bak interaction. Moreover, the individual and combined roles of Bax and Bak in S1-induced apoptosis were revealed. Our results showed that S1 induced a Bak-mediated apoptosis. Bak played a predominant role in either S1 or ABT-737-induced apoptosis through the cooperation with Bax on the formation of large oligomers on mitochondrial membrane.

Mutations of small heat shock proteins and human congenital diseases

The structure and properties of different members of a large family of small heat shock proteins (sHsp) playing an important role in cell homeostasis are described. Participation of the N-terminal domain in formation of large oligomers and chaperone activity of sHsp is analyzed. The structure of the α-crystallin domain of sHsp is characterized and the role of this domain in sHsp dimerization and chaperone activity is discussed. The properties of the C-terminal region of sHsp are described, and its participation in formation of large oligomers and chaperone activity are analyzed. The data from the literature on HspB1 and HspB3 mutations are presented, and involvement of these mutations in development of certain neurodegenerative diseases is discussed. Mutations of HspB4 are described and data on involvement of these mutations in development of cataract are presented. Multiple effects of HspB5 mutations are analyzed, and data are presented indicating that mutations of this protein are accompanied by development of different congenital diseases, such as cataract and different types of myopathies. The data on HspB6 and HspB8 mutations are presented, and feasible effects of these mutations on proteins structure are analyzed. Probable mechanisms underlying sHsp mutation-induced development of different congenital diseases are discussed.

Improved isolation of proteins tagged with glutathione S-transferase

A common affinity tag used to express and purify fusion proteins is glutathione S-transferase. However, many researchers have reported difficulty eluting GST-tagged proteins from the affinity matrix. This report demonstrates that the problem likely is due to the propensity of glutathione S-transferase to dimerize combined with a propensity of the tagged protein to oligomerize, which results in formation of large oligomers of fusion protein that are chelated by the affinity matrix. The solution to the problem is to use S-butylglutathione instead of glutathione to elute, as S-butylglutathione binds more tightly to glutathione S-transferase and overcomes the chelate effect. Moreover, in contrast to glutathione, S-butylglutathione has no reducing capability that might inactivate a tagged protein.

CABYR isoforms expressed in late steps of spermiogenesis bind with AKAPs and ropporin in mouse sperm fibrous sheath

BackgroundCABYR is a polymorphic calcium-binding protein of the sperm fibrous sheath (FS) which gene contains two coding regions (CR-A and CR-B) and is tyrosine as well as serine/threonine phosphorylated during in vitro sperm capacitation. Thus far, the detailed information on CABYR protein expression in mouse spermatogenesis is lacking. Moreover, because of the complexity of this polymorphic protein, there are no data on how CABYR isoforms associate and assemble into the FS.MethodsThe capacity of mouse CABYR isoforms to associate into dimers and oligomers, and the relationships between CABYR and other FS proteins were studied by gel electrophoresis, Western blotting, immunofluorescence, immunoprecipitation and yeast two-hybrid analyses.ResultsThe predominant form of mouse CABYR in the FS is an 80 kDa variant that contains only CABYR-A encoded by coding region A. CABYR isoforms form dimers by combining the 80 kDa CABYR-A-only variant with the 50 kDa variant that contains both CABYR-A and CABYR-B encoded by full length or truncated coding region A and B. It is proposed that this step is followed by the formation of larger oligomers, which then participate in the formation of the supramolecular structure of the FS in mouse sperm. The initial expression of CABYR occurs in the cytoplasm of spermatids at step 11 of spermiogenesis and increases progressively during steps 12-15. CABYR protein gradually migrates into the sperm flagellum and localizes to the FS of the principal piece during steps 15-16. Deletion of the CABYR RII domain abolished the interaction between CABYR and AKAP3/AKAP4 but did not abolish the interaction between CABYR and ropporin suggesting that CABYR binds to AKAP3/AKAP4 by its RII domain but binds to ropporin through another as yet undefined region.ConclusionsCABYR expresses at the late stage of spermiogenesis and its isoforms oligomerize and bind with AKAPs and ropporin. These interactions strongly suggest that CABYR participates in the assembly of complexes in the FS, which may be related to calcium signaling.

Visualization of the Molecular Dynamics of Lipopolysaccharide on the Plasma Membrane of Murine Macrophages by Total Internal Reflection Fluorescence Microscopy

The molecular action of Alexa 594-labeled lipopolysaccharide (LPS) from Escherichia coli was examined on living peritoneal macrophages of C57BL/6 mice by total internal reflection fluorescence microscope (TIRFM), and the molecular kinetics of LPS was analyzed. TIRFM visualization of the action of fluorescence-labeled LPS revealed an increase in the mean fluorescence intensity of LPS on the plasma membrane of wild type macrophages at 60 min after administration, indicating the oligomerization of LPS after binding to the macrophages. Additionally, a time-dependent sharp decrease in the mean diffusion coefficient of LPS was observed. On the other hand, both mean fluorescence intensity and diffusion coefficient of LPS in cases of TLR4(-/-), MD2(-/-), MyD88(-/-), and TRIF(-/-) macrophages were significantly different from the corresponding values of wild type macrophage, whereas differences were also noticed among these molecule-deficient macrophages. Furthermore, statistical analysis indicated the major role of receptors (TLR4 and MD2) and intracellular signaling molecules (MyD88 and TRIF) in oligomerization and lowering of the diffusion rate of LPS on the plasma membrane of murine macrophages, respectively.

HSPB2/MKBP, a novel and unique member of the small heat‐shock protein family

Although proteins belonging to the sHSP superfamily are diverse in sequence and size, most share characteristic features, including 1) a small molecular mass of 12-43 kDa, 2) a conserved alpha-crystallin domain of 80-100 residues, 3) formation of large oligomers, 4) a dynamic quaternary structure, and 5) induction by stress conditions and chaperone activity in suppressing protein aggregation. HSPB2/MKBP (myotonic dystrophy kinase-bind-protein) retains the structural motif of the alpha-crystallin family of HSPs but shows a unique nature compared with canonical family members, characterized by gene allocation, specific binding partners in skeletal muscle, and unique stress responsiveness. MKBP may be involved in the pathogenesis of myotonic dystrophy and contribute to the neuropathology in both Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis, Dutch type.

A study of the enzymatic oligomerization of 5-hydroxytryptamine using matrix-assisted laser desorption/ionization mass spectrometry

The role of tyrosinase in melanogenesis from 5-hydroxytryptamine was investigated by drawing samples from the reaction environment at different reaction times. Samples were ultrafiltered to remove the enzyme and lyophylized. In order to evaluate the role of non-enzymatic oxidation, a portion of the ultrafiltered samples was left to react under an oxygen stream for 24 hours prior to lyophilization. All the samples were analyzed by matrix-assisted laser desorption/ionization mass spectrometry. The formation of protonated tetramers was observed for samples immediately lyophilized, while further non-enzymatic reaction with bubbling oxygen led to formation of larger oligomers up to octamers. Clear analogies were observed between the data obtained on the enzymatic reaction and those reported in the literature on the electrochemically induced oxidation of 5-hydroxytryptamine. © 1997 John Wiley & Sons, Ltd.

Retention of p63 in an ER-Golgi intermediate compartment depends on the presence of all three of its domains and on its ability to form oligomers.

The type II membrane protein p63 is a resident protein of a membrane network interposed between rough ER and Golgi apparatus. To study the retention of p63, mutant forms were expressed in COS cells and the intracellular distribution determined by immunofluorescence microscopy. Investigation of chimeric constructs between p63 and the plasma membrane protein dipeptidylpeptidase IV showed that protein sequences from all three domains of the p63 protein are required to achieve complete intracellular retention. Mutational analysis of the 106-amino acid cytoplasmic tail of p63 revealed that the NH2-terminal 23 amino acids are necessary for retention. When p63 was solubilized with Triton X-100 and subjected to centrifugation at 100,000 g, it formed large, insoluble oligomers, particularly at neutral pH and below. A comparison of the behavior of wildtype and mutant p63 proteins in this assay revealed a perfect correlation between the formation of large oligomers and correct intracellular retention. These results suggest that self-association may be a major mechanism by which p63 is retained between the rough ER and the Golgi apparatus.