Kentaro Noi

High-speed atomic force microscopic observation of ATP-dependent rotation of the AAA+ chaperone p97

p97 (also called VCP and CDC-48) is an AAA+ chaperone, which consists of a substrate/cofactor-binding N domain and two ATPase domains (D1 and D2), and forms a homo-hexameric ring. p97 plays crucial roles in a variety of cellular processes such as the ubiquitin-proteasome pathway, the endoplasmic reticulum-associated protein degradation, autophagy, and modulation of protein aggregates. Mutations in human p97 homolog VCP are linked to neurodegenerative diseases. The key mechanism of p97 in these various functions has been proposed to be the disassembly of protein complexes. To understand the molecular mechanism of p97, we studied the conformational changes of hexameric CDC-48.1, a Caenorhabditis elegans p97 homolog, using high-speed atomic force microscopy. In the presence of ATP, the N-D1 ring repeatedly rotates ~23 ± 8° clockwise and resets relative to the D2 ring. Mutational analysis reveals that this rotation is induced by ATP binding to the D2 domain.

Nucleus factory on cavitation bubble for amyloid β fibril.

Structural evolution from monomer to fibril of amyloid β peptide is related to pathogenic mechanism of Alzheimer disease, and its acceleration is a long-running problem in drug development. This study reveals that ultrasonic cavitation bubbles behave as catalysts for nucleation of the peptide: The nucleation reaction is highly dependent on frequency and pressure of acoustic wave, and we discover an optimum acoustical condition, at which the reaction-rate constant for nucleation is increased by three-orders-of magnitudes. A theoretical model is proposed for explaining highly frequency and pressure dependent nucleation reaction, where monomers are captured on the bubble surface during its growth and highly condensed by subsequent bubble collapse, so that they are transiently exposed to high temperatures. Thus, the dual effects of local condensation and local heating contribute to dramatically enhance the nucleation reaction. Our model consistently reproduces the frequency and pressure dependences, supporting its essential applicability.

Ultrafast propagation of β-amyloid fibrils in oligomeric cloud.

Interaction between monomer peptides and seeds is essential for clarifying the fibrillation mechanism of amyloid β (Aβ) peptides. We monitored the deposition reaction of Aβ1–40 peptides on immobilized seeds grown from Aβ1–42, which caused formation of oligomers in the early stage. The deposition reaction and fibrillation procedure were monitored throughout by novel total-internal-reflection-fluorescence microscopy with a quartz-crystal microbalance (TIRFM-QCM) system. This system allows simultaneous evaluation of the amount of deposited peptides on the surface seeds by QCM and fibril nucleation and elongation by TIRFM. Most fibrils reached other nuclei, forming the fibril network across the nucleus hubs in a short time. We found a fibril-elongation rate two-orders-of-magnitude higher in an oligomeric cloud than reported values, indicating ultrafast transition of oligomers into fibrils.

Microtubule Severing by Katanin p60 AAA+ ATPase Requires the C-terminal Acidic Tails of Both α- and β-Tubulins and Basic Amino Acid Residues in the AAA+ Ring Pore

Background: Katanin p60 is a protein that actively severs microtubules. Results: Mutations within the AAA+ pore of katanin p60 and in the C-terminal regions of tubulins perturb efficient microtubule severing. Conclusion: Interactions between the conserved residues in the katanin p60 pore and the acidic tails of both tubulins may be important. Significance: Both tubulin molecules are essential for microtubule severing by katanin. The microtubule (MT) network is highly dynamic and undergoes dramatic reorganizations during the cell cycle. Dimers of α- and β-tubulins rapidly polymerize to and depolymerize from the end of MT fibrils in an intrinsic GTP-dependent manner. MT severing by ATP-driven enzymes such as katanin and spastin contributes significantly to microtubule dynamics, and it has been shown that katanin p60, a AAA+ family protein, has ATPase and MT-severing activities. The mechanism of MT severing by katanin p60 is poorly understood, and the residues in katanin p60 and tubulins important for severing activity were therefore explored in this study. MT-severing activity, but not ATPase activity, was inhibited by mutations of the conserved aromatic residue and the flanking basic residues in the pore region of the katanin p60 hexameric ring. When the acidic residue-rich C-terminal unstructured segment of either α- or β-tubulin was removed, polymerized MTs were resistant to katanin p60 treatment. Interactions between katanin p60 and the mutant MTs, on the other hand, were unaffected. Taken together, these findings led us to propose that the interactions between the positively charged residues of katanin p60 and the acidic tails of both tubulins are essential for efficient severing of MTs.

Functional Characterization of the Recombinant Group II Chaperonin α from Thermoplasma acidophilum

The functional characteristics of group II chaperonins, especially those from archaea, have not been elucidated extensively. Here, we performed a detailed functional characterization of recombinant chaperonin alpha subunits (16-mer) (Ta-cpn alpha) from the thermophilic archaea Thermoplasma acidophilum as a model protein of archaeal group II chaperonins. Recombinant Ta-cpn alpha formed an oligomeric ring structure similar to that of native protein, and displayed an ATP hydrolysis activity (optimal temperature: 60 degrees C) in the presence of either magnesium, manganese or cobalt ions. Ta-cpn alpha was able to bind refolding intermediates of Thermus MDH and GFP in the absence of ATP, and to promote the refolding of Thermus MDH at 50 degrees C in the presence of Mg2+-, Mn2+-, or Co2+-ATP. Ta-cpn alpha also prevented thermal aggregation of rhodanese and luciferase at 50 degrees C. Interestingly, Ta-cpn alpha in the presence of Mn2+ ion showed an increased hydrophobicity, which correlated with an increased efficiency in substrate protein binding. Our finding that Ta-cpn alpha chaperonin system displays folding assistance ability with ATP-dependent substrate release may provide a detailed look at the potential functional capabilities of archaeal chaperonins.

A potentially versatile nucleotide hydrolysis activity of group II chaperonin monomers from Thermoplasma acidophilum.

Compared to the group I chaperonins such as Escherichia coli GroEL, which facilitate protein folding, many aspects of the functional mechanism of archaeal group II chaperonins are still unclear. Here, we show that monomeric forms of archaeal group II chaperonin alpha and beta from Thermoplasma acidophilum may be purified stably and that these monomers display a strong AMPase activity in the presence of divalent ions, especially Co(2+) ion, in addition to ATPase and ADPase activities. Furthermore, other nucleoside phosphates (guanosine, cytidine, uridine, and inosine phosphates) in addition to adenine nucleotides were hydrolyzed. From analyses of the products of hydrolysis using HPLC, it was revealed that the monomeric chaperonin successively hydrolyzed the phosphoanhydride and phosphoester bonds of ATP in the order of gamma to alpha. This activity was strongly suppressed by point mutation of specific essential aspartic acid residues. Although these archaeal monomeric chaperonins did not alter the refolding of MDH, their novel versatile nucleotide hydrolysis activity might fulfill a new function. Western blot experiments demonstrated that the monomeric chaperonin subunits were also present in lysed cell extracts of T. acidophilum, and partially purified native monomer displayed Co(2+)-dependent AMPase activity.

The Highly Dynamic Nature of ERdj5 Is Key to Efficient Elimination of Aberrant Protein Oligomers through ER-Associated Degradation

ERdj5, composed of an N-terminal J domain followed by six thioredoxin-like domains, is the largest protein disulfide isomerase family member and functions as an ER-localized disulfide reductase that enhances ER-associated degradation (ERAD). Our previous studies indicated that ERdj5 comprises two regions, the N- and C-terminal clusters, separated by a linker loop and with distinct functional roles in ERAD. We here present a new crystal structure of ERdj5 with a largely different cluster arrangement relative to that in the original crystal structure. Single-molecule observation by high-speed atomic force microscopy visualized rapid cluster movement around the flexible linker loop, indicating the highly dynamic nature of ERdj5 in solution. ERdj5 mutants with a fixed-cluster orientation compromised the ERAD enhancement activity, likely because of less-efficient reduction of aberrantly formed disulfide bonds and prevented substrate transfer in the ERdj5-mediated ERAD pathway. We propose a significant role of ERdj5 conformational dynamics in ERAD of disulfide-linked oligomers.

A newly isolated Pex7-binding, atypical PTS2 protein P7BP2 is a novel dynein-type AAA+ protein

&NA; A newly isolated binding protein of peroxisomal targeting signal type 2 (PTS2) receptor Pex7, termed P7BP2, is transported into peroxisomes by binding to the longer isoform of Pex5p, Pex5pL, via Pex7p. The binding to Pex7p and peroxisomal localization of P7BP2 depends on the cleavable PTS2 in the N‐terminal region, suggesting that P7BP2 is a new PTS2 protein. By search on human database, three AAA+ domains are found in the N‐terminal half of P7BP2. Protein sequence alignment and motif search reveal that in the C‐terminal region P7BP2 contains additional structural domains featuring weak but sufficient homology to AAA+ domain. P7BP2 behaves as a monomer in gel‐filtration chromatography and the single molecule observed under atomic force microscope shapes a disc‐like ring. Collectively, these results suggest that P7BP2 is a novel dynein‐type AAA+ family protein, of which domains are arranged into a pseudo‐hexameric ring structure.

Expression, Functional Characterization, and Preliminary Crystallization of the Cochaperone Prefoldin from the Thermophilic Fungus Chaetomium thermophilum

Prefoldin is a hexameric molecular chaperone found in the cytosol of archaea and eukaryotes. Its hexameric complex is built from two related classes of subunits, and has the appearance of a jellyfish: Its body consists of a double β-barrel assembly with six long tentacle-like coiled coils protruding from it. Using the tentacles, prefoldin captures an unfolded protein substrate and transfers it to a group II chaperonin. Based on structural information from archaeal prefoldins, mechanisms of substrate recognition and prefoldin-chaperonin cooperation have been investigated. In contrast, the structure and mechanisms of eukaryotic prefoldins remain unknown. In this study, we succeeded in obtaining recombinant prefoldin from a thermophilic fungus, Chaetomium thermophilum (CtPFD). The recombinant CtPFD could not protect citrate synthase from thermal aggregation. However, CtPFD formed a complex with actin from chicken muscle and tubulin from porcine brain, suggesting substrate specificity. We succeeded in observing the complex formation of CtPFD and the group II chaperonin of C. thermophilum (CtCCT) by atomic force microscopy and electron microscopy. These interaction kinetics were analyzed by surface plasmon resonance using Biacore. Finally, we have shown the transfer of actin from CtPFD to CtCCT. The study of the folding pathway formed by CtPFD and CtCCT should provide important information on mechanisms of the eukaryotic prefoldin–chaperonin system.

Production of Single-Chain Fv Antibodies Specific for GA-Pyridine, an Advanced Glycation End-Product (AGE), with Reduced Inter-Domain Motion

Due to their lower production cost compared with monoclonal antibodies, single-chain variable fragments (scFvs) have potential for use in several applications, such as for diagnosis and treatment of a range of diseases, and as sensor elements. However, the usefulness of scFvs is limited by inhomogeneity through the formation of dimers, trimers, and larger oligomers. The scFv protein is assumed to be in equilibrium between the closed and open states formed by assembly or disassembly of VH and VL domains. Therefore, the production of an scFv with equilibrium biased to the closed state would be critical to overcome the problem in inhomogeneity of scFv for industrial or therapeutic applications. In this study, we obtained scFv clones stable against GA-pyridine, an advanced glycation end-product (AGE), by using a combination of a phage display system and random mutagenesis. Executing the bio-panning at 37 °C markedly improved the stability of scFvs. We further evaluated the radius of gyration by small-angle X-ray scattering (SAXS), obtained compact clones, and also visualized open–close dynamics of these scFvs by high-speed atomic force microscopy (HS-AFM), revealing that one of the compact clones was biased to the closed state. Finally, nuclear magnetic resonance (NMR) analysis revealed that peak intensity and line width became homogeneous, supporting that dynamic features and/or formation of oligomers was improved in the thus-obtained clone. These findings should contribute to the future industrial and therapeutic use of scFvs.