Distinct roles and actions of protein disulfide isomerase family enzymes in catalysis of nascent-chain disulfide bond formation.

The mammalian endoplasmic reticulum (ER) harbors more than 20 members of the protein disulfide isomerase (PDI) family that act to maintain proteostasis. Herein, we developed an in vitro system for directly monitoring PDI- or ERp46-catalyzed disulfide bond formation in ribosome-associated nascent chains of human serum albumin. The results indicated that ERp46 more efficiently introduced disulfide bonds into nascent chains with a short segment exposed outside the ribosome exit site than PDI. Single-molecule analysis by high-speed atomic force microscopy further revealed that PDI binds nascent chains persistently, forming a stable face-to-face homodimer, whereas ERp46 binds for a shorter time in monomeric form, indicating their different mechanisms for substrate recognition and disulfide bond introduction. Thus, ERp46 serves as a more potent disulfide introducer especially during the early stages of translation, whereas PDI can catalyze disulfide formation when longer nascent chains emerge out from ribosome.

Split conformation of Chaetomium thermophilum Hsp104 disaggregase.

Hsp104 and its bacterial homolog ClpB form hexameric ring structures and mediate protein disaggregation. The disaggregated polypeptide is thought to thread through the central channel of the ring. However, the dynamic behavior of Hsp104 during disaggregation remains unclear. Here, we reported the stochastic conformational dynamics and a split conformation of Hsp104 disaggregase from Chaetomium thermophilum (CtHsp104) in the presence of ADP by X-ray crystallography, cryo-electron microscopy (EM), and high-speed atomic force microscopy (AFM). ADP-bound CtHsp104 assembles into a 65 left-handed spiral filament in the crystal structure at a resolution of 2.7 Å. The unit of the filament is a hexamer of the split spiral structure. In the cryo-EM images, staggered and split hexameric rings were observed. Further, high-speed AFM observations showed that a substrate addition enhanced the conformational change and increased the split structure's frequency. Our data suggest that split conformation is an off-pathway state of CtHsp104 during disaggregation.

Hierarchical Model for the Role of J-Domain Proteins in Distinct Cellular Functions.

In Escherichia coli, the major bacterial Hsp70 system consists of DnaK, three J-domain proteins (JDPs: DnaJ, CbpA, and DjlA), and nucleotide exchange factor GrpE. JDPs determine substrate specificity for the Hsp70 system; however, knowledge on their specific role in bacterial cellular functions is limited. In this study, we demonstrated the role of JDPs in bacterial survival during heat stress and the DnaK-regulated formation of curli-extracellular amyloid fibers involved in biofilm formation. Genetic analysis demonstrate that only DnaJ is essential for survival at high temperature. On the other hand, either DnaJ or CbpA, but not DjlA, is sufficient to activate DnaK in curli production. Additionally, several DnaK mutants with reduced activity are able to complement the loss of curli production in E. coli ΔdnaK, whereas they do not recover the growth defect of the mutant strain at high temperature. Biochemical analyses reveal that DnaJ and CbpA are involved in the expression of the master regulator CsgD through the solubilization of MlrA, a DNA-binding transcriptional activator for the csgD promoter. Furthermore, DnaJ and CbpA also keep CsgA in a translocation-competent state by preventing its aggregation in the cytoplasm. Our findings support a hierarchical model wherein the role of JDPs in the Hsp70 system differs according to individual cellular functions.

The Cdc48-20S proteasome degrades a class of endogenous proteins in a ubiquitin-independent manner.

The 26S proteasome is the major degradation machinery for soluble proteins in eukaryotes. Recent evidence reveals the existence of an alternative ATP-powered protein degradation complex, the Cdc48-20S proteasome complex, and we have identified yeast Sod1, a copper-zinc superoxide dismutase, as an endogenous substrate protein. Here, we identified yeast Ths1, an essential threonyl tRNA synthetase, as another endogenous substrate protein of the Cdc48-20S proteasome. In order to analyze the degradation mechanism in more details, we established an in vitro degradation system reconstituted using purified yeast components. Recombinant Sod1 and Ths1 directly interacted with Cdc48, and were degraded in a Cdc48-20S proteasome-dependent manner. Because the substrate proteins were purified from E. coli cells, no eukaryotic modifications including ubiquitination and phosphorylation exist. Therefore, although the 26S proteasome requires ubiquitination for specific recognition of the substrate proteins, the Cdc48-20S proteasome can degrade a class of substrate proteins without any modifications.

A AAA ATPase Cdc48 with a cofactor Ubx2 facilitates ubiquitylation of a mitochondrial fusion-promoting factor Fzo1 for proteasomal degradation.

Dynamic functionality of mitochondria is maintained by continual fusion and fission events. A mitochondrial outer membrane protein Fzo1 plays a pivotal role upon mitochondrial fusion by homo-oligomerization to tether fusing mitochondria. Fzo1 is tightly regulated by ubiquitylations and the ubiquitin-responsible AAA protein Cdc48. Here, we show that a Cdc48 cofactor Ubx2 facilitates Fzo1 turnover. The Cdc48-Ubx2 complex has been shown to facilitate degradation of ubiquitylated proteins stacked at the protein translocation complex in the mitochondrial outer membrane by releasing them from the translocase. By contrast, in the degradation process of Fzo1, the Cdc48-Ubx2 complex appears to facilitate the degradation-signalling ubiquitylation of the substrate itself. In addition, the Cdc48-Ubx2 complex interacts with Ubp2, a deubiquitylase reversing the degradation-signalling ubiquitylation of Fzo1. These results suggest that the Cdc48-Ubx2 complex regulates Fzo1 turnover by modulating ubiquitylation status of the substrate.

Cyclization of Single-Chain Fv Antibodies Markedly Suppressed Their Characteristic Aggregation Mediated by Inter-Chain VH-VL Interactions.

Single-chain Fv (scFv) antibodies are recombinant proteins in which the variable regions of the heavy chain (VH) and light chain (VL) are connected by a short flexible polypeptide linker. ScFvs have the advantages of easy genetic manipulation and low-cost production using Escherichia coli compared with monoclonal antibodies, and are thus expected to be utilized as next-generation medical antibodies. However, the practical use of scFvs has been limited due to low homogeneity caused by their aggregation propensity mediated by inter-chain VH-VL interactions. Because the interactions between the VH and VL domains of antibodies are generally weak, individual scFvs are assumed to be in equilibrium between a closed state and an open state, in which the VH and VL domains are assembled and disassembled, respectively. This dynamic feature of scFvs triggers the formation of dimer, trimer, and larger aggregates caused by the inter-chain VH-VL interactions. To overcome this problem, the N-terminus and C-terminus were herein connected by sortase A-mediated ligation to produce a cyclic scFv. Open-closed dynamics and aggregation were markedly suppressed in the cyclic scFv, as judged from dynamic light scattering and high-speed atomic force microscopy analyses. Surface plasmon resonance and differential scanning fluorometry analysis revealed that neither the affinity for antigen nor the thermal stability was disrupted by the scFv cyclization. Generality was confirmed by applying the present method to several scFv proteins. Based on these results, cyclic scFvs are expected to be widely utilized in industrial and therapeutic applications.

Dynamic assembly of protein disulfide isomerase in catalysis of oxidative folding.

Time-resolved direct observations of proteins in action provide essential mechanistic insights into biological processes. Here, we present mechanisms of action of protein disulfide isomerase (PDI)-the most versatile disulfide-introducing enzyme in the endoplasmic reticulum-during the catalysis of oxidative protein folding. Single-molecule analysis by high-speed atomic force microscopy revealed that oxidized PDI is in rapid equilibrium between open and closed conformations, whereas reduced PDI is maintained in the closed state. In the presence of unfolded substrates, oxidized PDI, but not reduced PDI, assembles to form a face-to-face dimer, creating a central hydrophobic cavity with multiple redox-active sites, where substrates are likely accommodated to undergo accelerated oxidative folding. Such PDI dimers are diverse in shape and have different lifetimes depending on substrates. To effectively guide proper oxidative protein folding, PDI regulates conformational dynamics and oligomeric states in accordance with its own redox state and the configurations or folding states of substrates.

Functional characterization of UBXN-6, a C-terminal cofactor of CDC-48, in C. elegans.

CDC-48 is a AAA (ATPases associated with diverse cellular activities) chaperone and participates in a wide range of cellular activities. Its functional diversity is determined by differential binding of a variety of cofactors. In this study, we analyzed the physiological role of a CDC-48 cofactor UBXN-6 in Caenorhabditis elegans. The amount of UBXN-6 was markedly increased upon starvation, but not with the treatment of tunicamycin and rapamycin. The induction upon starvation is a unique characteristic for UBXN-6 among C-terminal cofactors of CDC-48. During starvation, lysosomal activity is triggered for rapid clearance of cellular materials. We observed the lysosomal activity by monitoring GLO-1::GFP, a marker for lysosome-related organelles. We found that more puncta of GLO-1::GFP were observed in the ubxn-6 deletion mutant after 12 h starvation compared with the wild-type strain. Taken together, we propose that UBXN-6 is involved in clearance of cellular materials upon starvation in C. elegans.

Development of transgenic Caenorhabditis elegans expressing human transthyretin as a model for drug screening.

Familial amyloid polyneuropathy is a hereditary systemic amyloidosis caused by a mutation in the transthyretin (TTR) gene. Amyloid deposits in tissues of patients contain not only full-length TTR but also C-terminal TTR fragments. However, in vivo models to evaluate the pathogenicity of TTR fragments have not yet been developed. Here, we generated transgenic Caenorhabditis elegans strains expressing several types of TTR fragments or full-length TTR fused to enhanced green fluorescent protein in the body wall muscle cells and analyzed the phenotypes of the worms. The transgenic strain expressing residues 81-127 of TTR, which included the ß-strands F and H, formed aggregates and caused defective worm motility and a significantly shortened lifespan compared with other strains. These findings suggest that the C-terminal fragments of TTR may contribute to cytotoxicity of TTR amyloidosis in vivo. By using this C. elegans model system, we found that (-)-epigallocatechin-3-gallate, a major polyphenol in green tea, significantly inhibited the formation of aggregates, the defective motility, and the shortened lifespan caused by residues 81-127 of TTR. These results suggest that our newly developed C. elegans model system will be useful for in vivo pathological analyses of TTR amyloidosis as well as drug screening.

Multitasking of Hsp70 chaperone in the biogenesis of bacterial functional amyloids.

Biofilms are intricate communities of microorganisms embedded in a self-produced matrix of extracellular polymer, which provides microbes survival advantages in stressful environments and can cause chronic infections in humans. Curli are functional amyloids that assemble on the extracellular surface of enteric bacteria such as Escherichia coli during biofilm development and colonization. The molecular chaperone DnaK, a bacterial Hsp70 homologue, promotes curli biogenesis via unknown mechanism(s). Here we show that DnaK increases the expression of CsgA and CsgB-the major and minor structural components of curli, respectively-via a quantity and quality control of RpoS, a stationary phase-specific alternative sigma factor regulating bacterial transcription, and CsgD, the master transcriptional regulator of curli formation. DnaK also keeps CsgA and CsgB in a translocation-competent state by binding to their signal peptides prone to aggregation. Our findings suggest that DnaK controls the homoeostasis of curli biogenesis at multiple stages to organize the biofilm matrix.

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

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 ß-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.

Inhibitory effects of Myricetin derivatives on curli-dependent biofilm formation in Escherichia coli.

Biofilms are well-organised communities of microbes embedded in a self-produced extracellular matrix (e.g., curli amyloid fibers) and are associated with chronic infections. Therefore, development of anti-biofilm drugs is important to combat with these infections. Previously, we found that flavonol Myricetin inhibits curli-dependent biofilm formation by Escherichia coli (IC50 = 46.2 µM). In this study, we tested activities of seven Myricetin-derivatives to inhibit biofilm formation by E. coli K-12 in liquid culture. Among them, only Epigallocatechin gallate (EGCG), a major catechin in green tea, inhibited biofilm formation of K-12 (IC50 = 5.9 µM) more efficiently than Myricetin. Transmission electron microscopy and immunoblotting analyses demonstrated that EGCG prevented curli production by suppressing the expression of curli-related proteins. Quantitative RT-PCR analysis revealed that the transcripts of csgA, csgB, and csgD were significantly reduced in the presence of EGCG. Interestingly, the cellular level of RpoS, a stationary-phase specific alternative sigma factor, was reduced in the presence of EGCG, whereas the rpoS transcript was not affected. Antibiotic-chase experiments and genetic analyses revealed that EGCG accelerated RpoS degradation by ATP-dependent protease ClpXP in combination with its adaptor RssB. Collectively, these results provide significant insights into the development of drugs to treat chronic biofilm-associated infections.

Biological and Pathological Implications of an Alternative ATP-Powered Proteasomal Assembly With Cdc48 and the 20S Peptidase.

The ATP-powered protein degradation machinery plays essential roles in maintaining protein homeostasis in all organisms. Robust proteolytic activities are typically sequestered within protein complexes to avoid the fatal removal of essential proteins. Because the openings of proteolytic chambers are narrow, substrate proteins must undergo unfolding. AAA superfamily proteins (ATPases associated with diverse cellular activities) are mostly located at these openings and regulate protein degradation appropriately. The 26S proteasome, comprising 20S peptidase and 19S regulatory particles, is the major ATP-powered protein degradation machinery in eukaryotes. The 19S particles are composed of six AAA proteins and 13 regulatory proteins, and bind to both ends of a barrel-shaped proteolytic chamber formed by the 20S peptidase. Several recent studies have reported that another AAA protein, Cdc48, can replace the 19S particles to form an alternative ATP-powered proteasomal complex, i.e., the Cdc48-20S proteasome. This review focuses on our current knowledge of this alternative proteasome and its possible linkage to amyotrophic lateral sclerosis.

Two Cdc48 cofactors Ubp3 and Ubx2 regulate mitochondrial morphology and protein turnover.

Mitochondria continuously undergo coordinated fusion and fission during vegetative growth to keep their homogeneity and to remove damaged components. A cytosolic AAA ATPase, Cdc48, is implicated in the mitochondrial fusion event and turnover of a fusion-responsible GTPase in the mitochondrial outer membrane, Fzo1, suggesting a possible linkage of mitochondrial fusion and Fzo1 turnover. Here, we identified two Cdc48 cofactor proteins, Ubp3 and Ubx2, involving mitochondria regulation. In the absence of UBP3, mitochondrial fragmentation and aggregation were observed. The turnover of Fzo1 was not affected in Δubp3, but instead a deubiquitylase Ubp12 that removes fusion-required polyubiquitin chains from Fzo1 was stabilized. Thus, excess amount of Ubp12 may lead to mitochondrial fragmentation by removal of fusion-competent ubiquitylated Fzo1. In contrast, deletion of UBX2 perturbed disassembly of Fzo1 oligomers and their degradation without alteration of mitochondrial morphology. The UBX2 deletion led to destabilization of Ubp2 that negatively regulates Fzo1 turnover by removing degradation-signalling polyubiquitin chains, suggesting that Ubx2 would directly facilitate Fzo1 degradation. These results indicated that two different Cdc48-cofactor complexes independently regulate mitochondrial fusion and Fzo1 turnover.

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.

Deviation of the typical AAA substrate-threading pore prevents fatal protein degradation in yeast Cdc48.

Yeast Cdc48 is a well-conserved, essential chaperone of ATPases associated with diverse cellular activity (AAA) proteins, which recognizes substrate proteins and modulates their conformations to carry out many cellular processes. However, the fundamental mechanisms underlying the diverse pivotal roles of Cdc48 remain unknown. Almost all AAA proteins form a ring-shaped structure with a conserved aromatic amino acid residue that is essential for proper function. The threading mechanism hypothesis suggests that this residue guides the intrusion of substrate proteins into a narrow pore of the AAA ring, thereby becoming unfolded. By contrast, the aromatic residue in one of the two AAA rings of Cdc48 has been eliminated through evolution. Here, we show that artificial retrieval of this aromatic residue in Cdc48 is lethal, and essential features to support the threading mechanism are required to exhibit the lethal phenotype. In particular, genetic and biochemical analyses of the Cdc48 lethal mutant strongly suggested that when in complex with the 20S proteasome, essential proteins are abnormally forced to thread through the Cdc48 pore to become degraded, which was not detected in wild-type Cdc48. Thus, the widely applicable threading model is less effective for wild-type Cdc48; rather, Cdc48 might function predominantly through an as-yet-undetermined mechanism.

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.

Imaging of bacterial multicellular behaviour in biofilms in liquid by atmospheric scanning electron microscopy.

Biofilms are complex communities of microbes that attach to biotic or abiotic surfaces causing chronic infectious diseases. Within a biofilm, microbes are embedded in a self-produced soft extracellular matrix (ECM), which protects them from the host immune system and antibiotics. The nanoscale visualisation of delicate biofilms in liquid is challenging. Here, we develop atmospheric scanning electron microscopy (ASEM) to visualise Gram-positive and -negative bacterial biofilms immersed in aqueous solution. Biofilms cultured on electron-transparent film were directly imaged from below using the inverted SEM, allowing the formation of the region near the substrate to be studied at high resolution. We visualised intercellular nanostructures and the exocytosis of membrane vesicles, and linked the latter to the trafficking of cargos, including cytoplasmic proteins and the toxins hemolysin and coagulase. A thick dendritic nanotube network was observed between microbes, suggesting multicellular communication in biofilms. A universal immuno-labelling system was developed for biofilms and tested on various examples, including S. aureus biofilms. In the ECM, fine DNA and protein networks were visualised and the precise distribution of protein complexes was determined (e.g., straight curli, flagella, and excreted cytoplasmic molecular chaperones). Our observations provide structural insights into bacteria-substratum interactions, biofilm development and the internal microbe community.

Thioflavin T as a fluorescence probe for monitoring RNA metabolism at molecular and cellular levels.

The intrinsically stochastic dynamics of mRNA metabolism have important consequences on gene regulation and non-genetic cell-to-cell variability; however, no generally applicable methods exist for studying such stochastic processes quantitatively. Here, we describe the use of the amyloid-binding probe Thioflavin T (ThT) for monitoring RNA metabolism in vitro and in vivo. ThT fluoresced strongly in complex with bacterial total RNA than with genomic DNA. ThT bound purine oligoribonucleotides preferentially over pyrimidine oligoribonucleotides and oligodeoxyribonucleotides. This property enabled quantitative real-time monitoring of poly(A) synthesis and phosphorolysis by polyribonucleotide phosphorylase in vitro. Cellular analyses, in combination with genetic approaches and the transcription-inhibitor rifampicin treatment, demonstrated that ThT mainly stained mRNA in actively dividing Escherichia coli cells. ThT also facilitated mRNA metabolism profiling at the single-cell level in diverse bacteria. Furthermore, ThT can also be used to visualise transitions between non-persister and persister cell states, a phenomenon of isogenic subpopulations of antibiotic-sensitive bacteria that acquire tolerance to multiple antibiotics due to stochastically induced dormant states. Collectively, these results suggest that probing mRNA dynamics with ThT is a broadly applicable approach ranging from the molecular level to the single-cell level.