Steps Of Disassembly Research Articles

Molecular insights into the very early steps of Aβ1-42 pentameric protofibril disassembly by PGG: A molecular dynamics simulation study

Aβ peptide aggregates were the main targets to treat AD. Previous experimental study demonstrated that the 1, 2, 3, 4, 6-penta-O-galloyl-β-D-glucopyranose (PGG, containing 5 gallate moieties) dramatically destabilized the preformed Aβ fibril in vitro and in vivo. However, the mechanisms were still poorly understood. Herein, by performing molecular dynamics simulations, the detailed mechanisms of the very early steps of Aβ1-42 pentameric protofibril disassembly by PGG were explored. It was demonstrated that the binding of PGG molecules to Aβ1-42 pentameric protofibril disrupted the β-sheet structure, weakened the interactions between Chain-1 and Chain-2, and disturbed the Lys28-Ala42 salt bridges in Aβ1-42 pentameric protofibril. We found that the hydrophobic interactions, hydrogen bonds, pi-pi interactions and cation-pi interactions existed between PGG molecules and amino acid residues in the corner of L-shaped part (Phe4-Leu17) were conducive to the disaggregation of the close interactions between Chain-1 and Chain-2. Moreover, PGG molecules disrupted the Lys28-Ala42 salt bridges not only by the hydrogen bonds and cation-pi interactions with Lys28 but also through the interactions with C-terminal hydrophobic amino acid residues. We believed that our results could provide significant insights into the mechanisms of the very early steps of Aβ1-42 pentameric protofibril disassembly by PGG.

Unique and redundant functions of cytoplasmic actins and nonmuscle myosin II isoforms at epithelial junctions.

The integrity and functions of epithelial barriers depend on the formation of adherens junctions (AJs) and tight junctions (TJs). A characteristic feature of AJs and TJs is their association with the cortical cytoskeleton composed of actin filaments and nonmuscle myosin II (NM-II) motors. Mechanical forces generated by the actomyosin cytoskeleton are essential for junctional assembly, stability, and remodeling. Epithelial cells express two different actin proteins and three NM-II isoforms, all known to be associated with AJs and TJs. Despite their structural similarity, different actin and NM-II isoforms have distinct biochemical properties, cellular distribution, and functions. The diversity of epithelial actins and myosin motors could be essential for the regulation of different steps of junctional formation, maturation, and disassembly. This review focuses on the roles of actin and NM-II isoforms in controlling the integrity and barrier properties of various epithelia. We discuss the effects of the depletion of individual actin isoforms and NM-II motors on the assembly and barrier function of AJs and TJs in model epithelial monolayers in vitro. We also describe the functional consequences of either total or tissue-specific gene knockout of different actins and NM-II motors, with a focus on the development and integrity of different epithelia in vivo.

The disassembly of lipid droplets in Chlamydomonas.

Lipid droplets (LDs) are ubiquitous and specialized organelles in eukaryotic cells. Consisting of a triacylglycerol core surrounded by a monolayer of membrane lipids, LDs are decorated with proteins and have myriad functions, from carbon/energy storage to membrane lipid remodeling and signal transduction. The biogenesis and turnover of LDs are therefore tightly coordinated with cellular metabolic needs in a fluctuating environment. Lipid droplet turnover requires remodeling of the protein coat, lipolysis, autophagy and fatty acid β-oxidation. Several key components of these processes have been identified in Chlamydomonas (Chlamydomonas reinhardtii), including the major lipid droplet protein, a CXC-domain containing regulatory protein, the phosphatidylethanolamine-binding DTH1 (DELAYED IN TAG HYDROLYSIS1), two lipases and two enzymes involved in fatty acid β-oxidation. Here, we review LD turnover and discuss its physiological significance in Chlamydomonas, a major model green microalga in research on algal oil.

On Characteristics of Symbolic Execution in the Problem of Assessing the Quality of Obfuscating Transformations

Obfuscation is used to protect programs from analysis and reverse engineering. There are theoretically effective and resistant obfuscation methods, but most of them are not implemented in practice yet. The main reasons are large overhead for the execution of obfuscated code and the limitation of application only to a specific class of programs. On the other hand, a large number of obfuscation methods have been developed that are applied in practice. The existing approaches to the assessment of such obfuscation methods are based mainly on the static characteristics of programs. Therefore, the comprehensive (taking into account the dynamic characteristics of programs) justification of their effectiveness and resistance is a relevant task. It seems that such a justification can be made using machine learning methods, based on feature vectors that describe both static and dynamic characteristics of programs. In this paper, it is proposed to build such a vector on the basis of characteristics of two compared programs: the original and obfuscated, original and deobfuscated, obfuscated and deobfuscated. In order to obtain the dynamic characteristics of the program, a scheme based on a symbolic execution is constructed and presented in this paper. The choice of the symbolic execution is justified by the fact that such characteristics can describe the difficulty of comprehension of the program in dynamic analysis. The paper proposes two implementations of the scheme: extended and simplified. The extended scheme is closer to the process of analyzing a program by an analyst, since it includes the steps of disassembly and translation into intermediate code, while in the simplified scheme these steps are excluded. In order to identify the characteristics of symbolic execution that are suitable for assessing the effectiveness and resistance of obfuscation based on machine learning methods, experiments with the developed schemes were carried out. Based on the obtained results, a set of suitable characteristics is determined.

Release of Human TFIIB from Actively Transcribing Complexes Is Triggered upon Synthesis of 7- and 9-nt RNAs

RNA polymerase II (Pol II) and its general transcription factors assemble on the promoters of mRNA genes to form large macromolecular complexes that initiate transcription in a regulated manner. During early transcription, these complexes undergo dynamic rearrangement and disassembly as Pol II moves away from the start site of transcription and transitions into elongation. One step in disassembly is the release of the general transcription factor TFIIB, although the mechanism of release and its relationship to the activity of transcribing Pol II is not understood. We developed a single-molecule fluorescence transcription system to investigate TFIIB release in vitro. Leveraging our ability to distinguish active from inactive complexes, we found that nearly all transcriptionally active complexes release TFIIB during early transcription. Release is not dependent on the contacts TFIIB makes with its recognition element in promoter DNA. We identified two different points in early transcription at which release is triggered, reflecting heterogeneity across the population of actively transcribing complexes. TFIIB releases after both trigger points with similar kinetics, suggesting the rate of release is independent of the molecular transformations that prompt release. Together our data support the model that TFIIB release is important for Pol II to successfully escape the promoter as initiating complexes transition into elongation complexes.

Regulated changes in material properties underlie centrosome disassembly during mitotic exit.

Centrosomes must resist microtubule-mediated forces for mitotic chromosome segregation. During mitotic exit, however, centrosomes are deformed and fractured by those same forces, which is a key step in centrosome disassembly. How the functional material properties of centrosomes change throughout the cell cycle, and how they are molecularly tuned, remain unknown. Here, we used optically induced flow perturbations to determine the molecular basis of centrosome strength and ductility in C. elegans embryos. We found that both properties declined sharply at anaphase onset, long before natural disassembly. This mechanical transition required PP2A phosphatase and correlated with inactivation of PLK-1 (Polo kinase) and SPD-2 (Cep192). In vitro, PLK-1 and SPD-2 directly protected centrosome scaffolds from force-induced disassembly. Our results suggest that, before anaphase, PLK-1 and SPD-2 respectively confer strength and ductility to the centrosome scaffold so that it can resist microtubule-pulling forces. In anaphase, centrosomes lose PLK-1 and SPD-2 and transition to a weak, brittle state that enables force-mediated centrosome disassembly.

In Vitro Reconstitution Defines the Minimal Requirements for Cdc48-Dependent Disassembly of the CMG Helicase in Budding Yeast

SummaryDisassembly of the replisome is the final step of chromosome duplication in eukaryotes. In budding yeast and metazoa, cullin ubiquitin ligases are required to ubiquitylate the Cdc45-MCM-GINS (CMG) helicase that lies at the heart of the replisome, leading to a disassembly reaction that is dependent upon the ATPase known as Cdc48 or p97. Here, we describe the reconstitution of replisome disassembly, using a purified complex of the budding yeast replisome in association with the cullin ligase SCFDia2. Upon addition of E1 and E2 enzymes, together with ubiquitin and ATP, the CMG helicase is ubiquitylated on its Mcm7 subunit. Subsequent addition of Cdc48, together with its cofactors Ufd1-Npl4, drives efficient disassembly of ubiquitylated CMG, thereby recapitulating the steps of replisome disassembly that are observed in vivo. Our findings define the minimal requirements for disassembly of the eukaryotic replisome and provide a model system for studying the disassembly of protein complexes by Cdc48-Ufd1-Npl4.

Topological Analysis of Transthyretin Disassembly Mechanism: Surface-Induced Dissociation Reveals Hidden Reaction Pathways.

The proposed mechanism of fibril formation of transthyretin (TTR) involves self-assembly of partially unfolded monomers. However, the mechanism(s) of disassembly to monomer and potential intermediates involved in this process are not fully understood. In this study, native mass spectrometry and surface-induced dissociation (SID) are used to investigate the TTR disassembly mechanism(s) and the effects of temperature and ionic strength on the kinetics of TTR complex formation. Results from the SID of hybrid tetramers formed during subunit exchange provide strong evidence for a two-step mechanism whereby the tetramer dissociates to dimers that then dissociate to monomers. Also, the SID results uncovered a hidden pathway in which a specific topology of the hybrid tetramer is directly produced by assembly of dimers in the early steps of TTR disassembly. Implementation of SID to dissect protein topology during subunit exchange provides unique opportunities to gain unparalleled insight into disassembly pathways.

Inositol phosphates are assembly co-factors for HIV-1.

Information of the HIV-1 virus particle, a short, 14-amino acid segment called SP1, located in the Gag structural protein1, plays a critical role. During virus assembly the SP1 peptide and seven preceding residues fold into a six-helix bundle (6HB) that holds together the Gag hexamer and facilitates formation of a curved immature hexagonal lattice underneath the viral membrane2,3. Upon completion of assembly and budding, proteolytic cleavage of Gag leads to virus maturation, in which the immature lattice is broken down; the liberated CA domain of Gag then re-assembles into the mature conical capsid that encloses the viral genome and associated enzymes. Folding and proteolysis of the 6HB are critical rate-limiting steps of both Gag assembly and disassembly, and the 6HB is an established target of HIV-1 inhibitors4,5. Using a combination of structural and functional analyses, we show here that inositol hexakisphosphate (IP6) facilitates formation of the 6HB and assembly of the immature HIV-1 Gag lattice. IP6 makes ionic contacts with two rings of lysine residues at the center of the Gag hexamer. Proteolytic cleavage then unmasks an alternative binding site, where IP6 interaction promotes assembly of the mature capsid lattice. These studies identify IP6 as a naturally occurring small molecule that promotes both HIV-1 assembly and maturation.

Mitotic Disassembly of Nuclear Pore Complexes Involves CDK1- and PLK1-Mediated Phosphorylation of Key Interconnecting Nucleoporins

SummaryDuring interphase, the nuclear envelope (NE) serves as a selective barrier between cytosol and nucleoplasm. When vertebrate cells enter mitosis, the NE is dismantled in the process of nuclear envelope breakdown (NEBD). Disassembly of nuclear pore complexes (NPCs) is a key aspect of NEBD, required for NE permeabilization and formation of a cytoplasmic mitotic spindle. Here, we show that both CDK1 and polo-like kinase 1 (PLK1) support mitotic NPC disintegration by hyperphosphorylation of Nup98, the gatekeeper nucleoporin, and Nup53, a central nucleoporin linking the inner NPC scaffold to the pore membrane. Multisite phosphorylation of Nup53 critically contributes to its liberation from its partner nucleoporins, including the pore membrane protein NDC1. Initial steps of NPC disassembly in semi-permeabilized cells can be reconstituted by a cocktail of mitotic kinases including cyclinB-CDK1, NIMA, and PLK1, suggesting that the unzipping of nucleoporin interactions by protein phosphorylation is an important principle underlying mitotic NE permeabilization.

Cdk5 regulates developmental remodeling of mushroom body neurons in Drosophila.

During metamorphosis, axons and dendrites of the mushroom body (MB) in the Drosophila central brain are remodeled extensively to support the transition from larval to adult behaviors. We show here that the neuronal cyclin-dependent kinase, Cdk5, regulates the timing and rate of mushroom body remodeling: reduced Cdk5 activity causes a delay in pruning of MB neurites, while hyperactivation accelerates it. We further show that Cdk5 cooperates with the ubiquitin-proteasome system in this process. Finally, we show that Cdk5 modulates the first overt step in neurite disassembly, dissolution of the neuronal tubulin cytoskeleton, and provide evidence that it also acts at additional steps of MB pruning. These data show that Cdk5 regulates the onset and extent of remodeling of the Drosophila MB. Given the wide phylogenetic conservation of Cdk5, we suggest that it is likely to play a role in developmental remodeling in other systems, as well. Moreover, we speculate that the well-established role of Cdk5 in neurodegeneration may involve some of the same cellular mechanisms that it uses during developmental remodeling.

Photoreceptor ablation initiates the immediate loss of glutamate receptors in postsynaptic bipolar cells in retina.

Structural changes underlying neurodegenerative diseases include dismantling of synapses, degradation of circuitry, and even massive rewiring. Our limited understanding of synapse dismantling stems from the inability to control the timing and extent of cell death. In this study, selective ablation of cone photoreceptors in live mouse retina and tracking of postsynaptic partners at the cone-to-ON cone bipolar cell synapse reveals that early reaction to cone loss involves rapid and local changes in postsynaptic glutamate receptor distribution. Glutamate receptors disappear with a time constant of 2 h. Furthermore, binding of glutamate receptors by agonists and antagonists is insufficient to rescue glutamate receptor loss, suggesting that receptor allocation depends on the physical presence of cones. These findings demonstrate that the initial step in synapse disassembly involves postsynaptic receptor loss rather than dendritic retraction, providing insight into the early stages of neurodegenerative disease.

Structural Documentation of Glycan Epitopes: Sequential Mass Spectrometry and Spectral Matching

Documenting mass spectral data is a fundamental aspect of accepted protocols. In this report, we contrast MS(n) sequential disassembly spectra obtained from natural and synthetic glycan epitopes. The epitopes considered are clusters found on conjugate termini of lipids and N- and O-glycans of proteins. The latter are most frequently pendant through a CID-labile HexNAc glycosidic linkage. The synthetic samples were supplied by collaborating colleagues and commercial sources and usually possessed a readily released reducing-end linker, a by-product of synthesis. All samples were comparably methylated, extracted, and MS(n) disassembled to compare their linkage and branching spectral details. Both sample types provide B-ion type fragments early in a disassembly pathway and their compositions are a suggestion of structure. Further steps of disassembly are necessary to confirm the details of linkage and branching. Included in this study were various Lewis and H antigens, 3- and 6-linked sialyl-lactosamine, NeuAc-2,8-NeuAc dimer, and Galα1,3Gal. Sample infusion provided high quality spectral data whereas disassembly to small fragments generates reproducible high signal/noise spectra for spectral matching. All samples were analyzed as sodium adducted positive ions. This study includes comparability statistics and evaluations on several mass spectrometers.

Mitofusin Proteins Tether Proteoliposomes as Shown by Cryo-EM

A balance between mitochondrial membrane fusion and fission is required for normal mitochondrial morphology and function. Shifts in mitochondrial morphology as a result of changes in the balance of membrane fusion and fission accompany cellular differentiation and changes in metabolic state. It is thought that the severe, early-onset peripheral neuropathy Charcot-Marie-Tooth type 2A disease is a manifestation of mitochondrial fusion malfunction as a result of a mutation of one of the two human mitofusins, mitofusin 2. Our aim is to reveal the mechanism by which mitofusins cause membrane fusion. Both mammalian mitofusins contain a large GTPase domain at their N-terminus followed by two transmembrane domains and a short C-terminal domain. It is predicted that GTP hydrolysis and mitofusin conformational changes are coupled to membrane fusion. However, the precise mechanism for mitofusin-mediated membrane fusion remains an open question. In fact, it is still unknown if mitochondrial outer membrane fusion proceeds through the canonical steps of tethering, docking, fusion, and disassembly. To gain insight into the conformational changes that lead to membrane fusion we are examining the structure of mitofusins in a lipid bilayer. As two-pass transmembrane proteins, mitofusins have proven difficult to express and purify in their full-length state and are poor candidates for crystallography. Here we present a purification strategy and the first cryo-em images of full-length mitofusins in a lipid bilayer. In addition, we show that mitofusins tether proteolipsosomes by forming oligomers that interact in trans between synthetic membranes. This implies that mitofusins are sufficient for driving the first step of membrane fusion.

Possible steps of complete disassembly of post-termination complex by yeast eEF3 deduced from inhibition by translocation inhibitors

Ribosomes, after one round of translation, must be recycled so that the next round of translation can occur. Complete disassembly of post-termination ribosomal complex (PoTC) in yeast for the recycling consists of three reactions: release of tRNA, release of mRNA and splitting of ribosomes, catalyzed by eukaryotic elongation factor 3 (eEF3) and ATP. Here, we show that translocation inhibitors cycloheximide and lactimidomycin inhibited all three reactions. Cycloheximide is a non-competitive inhibitor of both eEF3 and ATP. The inhibition was observed regardless of the way PoTC was prepared with either release factors or puromycin. Paromomycin not only inhibited all three reactions but also re-associated yeast ribosomal subunits. On the other hand, sordarin or fusidic acid, when applied together with eEF2/GTP, specifically inhibited ribosome splitting without blocking of tRNA/mRNA release. From these inhibitor studies, we propose that, in accordance with eEF3’s known function in elongation, the release of tRNA via exit site occurs first, then mRNA is released, followed by the splitting of ribosomes during the disassembly of post-termination complexes catalyzed by eEF3 and ATP.

Mitotic lamin disassembly is triggered by lipid-mediated signaling

Disassembly of the nuclear lamina is a key step during open mitosis in higher eukaryotes. The activity of several kinases, including CDK1 (cyclin-dependent kinase 1) and protein kinase C (PKC), has been shown to trigger mitotic lamin disassembly, yet their precise contributions are unclear. In this study, we develop a quantitative imaging assay to study mitotic lamin B1 disassembly in living cells. We find that CDK1 and PKC act in concert to mediate phosphorylation-dependent lamin B1 disassembly during mitosis. Using ribonucleic acid interference (RNAi), we showed that diacylglycerol (DAG)-dependent PKCs triggered rate-limiting steps of lamin disassembly. RNAi-mediated depletion or chemical inhibition of lipins, enzymes that produce DAG, delayed lamin disassembly to a similar extent as does PKC inhibition/depletion. Furthermore, the delay of lamin B1 disassembly after lipin depletion could be rescued by the addition of DAG. These findings suggest that lipins activate a PKC-dependent pathway during mitotic lamin disassembly and provide evidence for a lipid-mediated mitotic signaling event.

Nucleosome Dynamics Studied by Single Molecule FRET

We studied the mechanism of nucleosome opening to allow DNA access, using single molecule FRET. Here we show evidence for a previously uncharacterized intermediate structural state that occurs before H2A-H2B dimer release, and is characterized by an increased distance between H2B and the nucleosomal dyad. Our data suggest that the first step in nucleosome disassembly is the opening of the (H3-H4)2 tetramer/(H2A-H2B) dimer interface, followed by H2A-H2B dimer release from the DNA and, lastly, (H3-H4)2 tetramer removal (see Figure). We estimate that the open intermediate state is populated at 0.2 - 3 % under physiological conditions, and could have significant in vivo implications for factor-mediated histone removal and exchange, as well as for regulating DNA accessibility to the transcription and replication machinery. Additionally, histone variants and histone modifications could substantially change the stability of the nucleosome. Our most recent spFRET data indicate that such effects may occur both at the level of DNA-histone and histone-histone interactions.

Nucleosome accessibility governed by the dimer/tetramer interface

Nucleosomes are multi-component macromolecular assemblies which present a formidable obstacle to enzymatic activities that require access to the DNA, e.g. DNA and RNA polymerases. The mechanism and pathway(s) by which nucleosomes disassemble to allow DNA access are not well understood. Here we present evidence from single molecule FRET experiments for a previously uncharacterized intermediate structural state before H2A–H2B dimer release, which is characterized by an increased distance between H2B and the nucleosomal dyad. This suggests that the first step in nucleosome disassembly is the opening of the (H3–H4)2 tetramer/(H2A–H2B) dimer interface, followed by H2A–H2B dimer release from the DNA and, lastly, (H3–H4)2 tetramer removal. We estimate that the open intermediate state is populated at 0.2–3% under physiological conditions. This finding could have significant in vivo implications for factor-mediated histone removal and exchange, as well as for regulating DNA accessibility to the transcription and replication machinery.

The three-dimensional structure of ryegrass mottle virus at 2.9 Å resolution

The crystal structure of the sobemovirus Ryegrass mottle virus (RGMoV) has been determined at 2.9 Å resolution. The coat protein has a canonical jellyroll β-sandwich fold. In comparison to other sobemoviruses the RGMoV coat protein is missing several residues in two of the loop regions. The first loop contributes to contacts between subunits around the quasi-threefold symmetry axis. The altered contact interface results in tilting of the subunits towards the quasi-threefold axis. The assembly of the T = 3 capsid of sobemoviruses is controlled by the N-termini of C subunits forming a so-called β-annulus. The other loop that is smaller in the RGMoV structure contains a helix that participates in stabilization of the β-annulus in other sobemoviruses. The loss of interaction between the RGMoV loop and the β-annulus has been compensated for by additional interactions between the N-terminal arms. As a consequence of these differences, the diameter of the RGMoV particle is 8 Å smaller than that of the other sobemoviruses. The interactions of coat proteins in sobemovirus capsids involve calcium ions. Depletion of calcium ions results in particle swelling, which is considered a first step in disassembly. We could not identify any density for metal ions in the proximity of the conserved residues normally involved in calcium binding, but the RGMoV structure does not show any signs of swelling. A likely reason is the low pH (3.0) of the crystallization buffer in which the groups interacting with the calcium ions are not charged.

An in vitro nuclear disassembly system reveals a role for the RanGTPase system and microtubule-dependent steps in nuclear envelope breakdown

During prophase, vertebrate cells disassemble their nuclear envelope (NE) in the process of NE breakdown (NEBD). We have established an in vitro assay that uses mitotic Xenopus laevis egg extracts and semipermeabilized somatic cells bearing a green fluorescent protein–tagged NE marker to study the molecular requirements underlying the dynamic changes of the NE during NEBD by live microscopy. We applied our in vitro system to analyze the role of the Ran guanosine triphosphatase (GTPase) system in NEBD. Our study shows that high levels of RanGTP affect the dynamics of late steps of NEBD in vitro. Also, inhibition of RanGTP production by RanT24N blocks the dynamic rupture of nuclei, suggesting that the local generation of RanGTP around chromatin may serve as a spatial cue in NEBD. Furthermore, the microtubule-depolymerizing drug nocodazole interferes with late steps of nuclear disassembly in vitro. High resolution live cell imaging reveals that microtubules are involved in the completion of NEBD in vivo by facilitating the efficient removal of membranes from chromatin.