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[Image: see text] The onset of neurodegenerative disorders (NDs), such as Alzheimer’s disease, is associated with the accumulation of aggregates of misfolded proteins. We previously showed that chemical knockdown of ND-related aggregation-prone proteins can be achieved by proteolysis targeting chimeras (PROTACs). However, hetero-bifunctional PROTACs generally show poor permeability into the central nervous system, where NDs are located. Here, we document the conversion of one of our PROTACs into hydrophobic tags (HyTs), another class of degraders bearing hydrophobic degrons. This conversion decreases the molecular weight and the number of hydrogen bond donors/acceptors. All the developed HyTs lowered the level of mutant huntingtin, an aggregation-prone protein, with potency comparable to that of the parent PROTAC. Through IAM chromatography analysis and in vivo brain penetration assay of the HyTs, we discovered a brain-permeable HyT. Our results and mechanistic analysis indicate that conversion of protein degraders into HyTs could be a useful approach to improve their drug-like properties.

Life depends on proteins, which all exist in nascent states when the growing polypeptide chain is covalently attached to a tRNA within the ribosome. Although the nascent chains; i.e., polypeptidyl-tRNAs (pep-tRNAs), are considered as merely transient intermediates during protein synthesis, recent advances have revealed that they are directly involved in a variety of cell functions, such as gene expression control. An increasing appreciation for fine-tuning at translational levels demands a general method to handle the pep-tRNAs on a large scale. Here, we developed a method termed peptidyl-tRNA enrichment using organic extraction and silica adsorption (PETEOS), and then identify their polypeptide moieties by mass spectrometry. As a proof-of-concept experiment using Escherichia coli, we identified ∼800 proteins derived from the pep-tRNAs, which were markedly biased towards the N-termini in the proteins, reflecting that PETEOS captured the intermediate pep-tRNA population during translation. Furthermore, we observed the changes in the pep-tRNA set in response to heat shock or antibiotic treatments. In summary, PETEOS will complement conventional methods for profiling nascent chains such as ribosome profiling.Significance StatementIn the central dogma of biology, RNA and protein are usually regarded as two completely independent molecular species. However, they are combined into a single species called peptidyl-tRNA (pep-tRNA) during the translation process in the ribosome. Despite the importance of pep-tRNAs as precursors of all proteins in the cell, a general method to analyze pep-tRNAs on a large scale was lacking. Taking advantage of the properties of pep-tRNAs as RNA and protein, we developed a method to enrich the pep-tRNAs by organic solvent extraction and silica column separation. The method, termed PETEOS, not only provides a unique approach to examine the nascent state of proteins but also may be effective in capturing snapshots of translation status in the cell.

Escherichia coli has multiple pathways to release nonproductive ribosome complexes stalled at the 3' end of nonstop mRNA: tmRNA (SsrA RNA)-mediated trans-translation and stop codon-independent termination by ArfA/RF2 or ArfB (YaeJ). The arfA mRNA lacks a stop codon and its expression is repressed by trans-translation. Therefore, ArfA is considered to complement the ribosome rescue activity of trans-translation, but the physiological situations in which ArfA is expressed have not been elucidated. Here, we found that the excision of CP4-57 prophage adjacent to E. coli ssrA leads to the inactivation of tmRNA and switches the primary rescue pathway from trans-translation to ArfA/RF2. This "rescue-switching" rearranges not only the proteome landscape in E. coli but also the phenotype such as motility. Furthermore, among the proteins with significantly increased abundance in the ArfA

Peptides with strong binding affinities for poly(methyl methacrylate) (PMMA) resin were designed by use of materials informatics technology based on molecular dynamics simulation for the purpose of covering the resin surface with adhesive peptides, which were expected to result in eco-friendly and biocompatible biomaterials. From the results of binding affinity obtained with this molecular simulation, it was confirmed that experimental values could be predicted with errors

The Escherichia coli chaperonin GroEL/ES (GroE) is one of the most extensively studied molecular chaperones. So far, ~80 proteins in E. coli are identified as GroE substrates that obligately require GroE for folding in vivo. In GroE-depleted cells, these substrates, when overexpressed, tend to form aggregates, whereas the GroE substrates expressed at low or endogenous levels are degraded, probably due to misfolded states. However, the protease(s) involved in the degradation process has not been identified. We conducted a mass-spectrometry-based proteomics approach to investigate the effects of three ATP-dependent proteases, Lon, ClpXP, and HslUV, on the E. coli proteomes under GroE-depleted conditions. A label-free quantitative proteomic method revealed that Lon protease is the dominant protease that degrades the obligate GroE substrates in the GroE-depleted cells. The deletion of DnaK/DnaJ, the other major E. coli chaperones, in the ∆lon strain did not cause major alterations in the expression or folding of the obligate GroE substrates, supporting the idea that the folding of these substrates is predominantly dependent on GroE.

Escherichia coli has multiple pathways to release a nonproductive ribosome complex stalled at the 3’ end of nonstop mRNAs: SsrA RNA-mediated trans-translation and stop codon-independent termination by ArfA/RF2 or ArfB (YaeJ). The arfA mRNA lacks a stop codon and thus its expression is repressed by trans-translation. Therefore, ArfA is considered to complement the ribosome rescue activity of trans-translation, but the situations in which ArfA is expressed to rescue the nonproductive complexes have not been elucidated. Here, we demonstrated that the excision of the CP4-57 prophage adjacent to the E. coli ssrA gene leads to the inactivation of SsrA RNA and switches the primary rescue pathway from trans-translation to the ArfA/RF2 pathway. A comparative quantitative proteomic analysis revealed that the switching of the rescue pathway rearranges not only the proteome landscape in E. coli cells but also the phenotype, such as motility. Among the proteins with significantly increased abundance in the ssrA-inactivated cells, we found ZntR, whose mRNA is transcribed together as the upstream part of the nonstop arfA mRNA. Further analysis revealed that the translation of the nonstop ORF of arfA triggered the repression of the upstream zntR ORF, via trans-translation-coupled exonucleolytic (RUTEX) degradation by a polynucleotide phosphorylase. These results provide a novel example of trans-translation-dependent regulation, and shed new light on the physiological roles of prophages in gene expression.

SecM, a bacterial secretion monitor protein, posttranscriptionally regulates downstream gene expression via translation elongation arrest. SecM contains a characteristic amino acid sequence called the arrest sequence at its C-terminus, and this sequence acts within the ribosomal exit tunnel to stop translation. It has been widely assumed that the arrest sequence within the ribosome tunnel is sufficient for translation arrest. We have previously shown that the nascent SecM chain outside the ribosomal exit tunnel stabilizes translation arrest, but the molecular mechanism is unknown. In this study, we found that residues 57–98 of the nascent SecM chain are responsible for stabilizing translation arrest. We performed alanine/serine-scanning mutagenesis of residues 57–98 to identify D79, Y80, W81, H84, R87, I90, R91, and F95 as the key residues responsible for stabilization. The residues were predicted to be located on and near an α-helix-forming segment. A striking feature of the α-helix is the presence of an arginine patch, which interacts with the negatively charged ribosomal surface. A photocross-linking experiment showed that Y80 is adjacent to the ribosomal protein L23, which is located next to the ribosomal exit tunnel when translation is arrested. Thus, the folded nascent SecM chain that emerges from the ribosome exit tunnel interacts with the outer surface of the ribosome to stabilize translation arrest.

Intracellular photocatalytic-proximity labeling (iPPL) was developed to profile protein-protein interactions in the microenvironment of living cells. Acriflavine was found to be an efficient cell-membrane-permeable photocatalyst for introduction into the genetically HaloTag-fused protein of interest for iPPL with a radical labeling reagent, 1-methyl-4-arylurazole. iPPL was applied to the histone-associated protein H2B in HaloTag-H2B expressing HEK293FT cells. The proteins directly interacting with histones and RNA-binding proteins were selectively labeled in the intracellular environment, suggesting that the iPPL method has a smaller labeling radius (CA. 6 nm) than the BioID and APEX methods.

Continuous translation elongation, irrespective of amino acid sequences, is a prerequisite for living organisms to produce their proteomes. However, nascent polypeptide products bear an inherent risk of elongation abortion. For example, negatively charged sequences with occasional intermittent prolines, termed intrinsic ribosome destabilization (IRD) sequences, weaken the translating ribosomal complex, causing certain nascent chain sequences to prematurely terminate translation. Here, we show that most potential IRD sequences in the middle of open reading frames remain cryptic and do not interrupt translation, due to two features of the nascent polypeptide. Firstly, the nascent polypeptide itself spans the exit tunnel, and secondly, its bulky amino acid residues occupy the tunnel entrance region, thereby serving as a bridge and protecting the large and small ribosomal subunits from dissociation. Thus, nascent polypeptide products have an inbuilt ability to ensure elongation continuity.

To understand the physiological role of NADPH-thioredoxin reductase C (NTRC) in cyanobacteria, we investigated an NTRC-deficient mutant strain of Anabaena sp., PCC 7120, cultivated under different regimes of nitrogen supplementation and light exposure. The deletion of ntrC did not induce a change in the cell structure and metabolic pathways. However, time-dependent changes in the abundance of specific proteins and metabolites were observed. A decrease in chlorophyll a was correlated with a decrease in chlorophyll a biosynthesis enzymes and photosystem I subunits. The deletion of ntrC led to a deregulation of nitrogen metabolism, including the NtcA accumulation and heterocyst-specific proteins while nitrate ions were available in the culture medium. Interestingly, this deletion resulted in a redox imbalance, indicated by higher peroxide levels, higher catalase activity and the induction of chaperones such as MsrA. Surprisingly, the antioxidant protein 2-CysPrx was downregulated. The deficiency in ntrC also resulted in the accumulation of metabolites such as 6-phosphogluconate, ADP and ATP. Higher levels of NADP+ and NADPH partly correlated with higher G6PDH activity. Rather than impacting protein expression levels, NTRC appears to be involved in the direct regulation of enzymes, especially during the dark-to-light transition period.

Nuclear import receptors (NIRs) not only transport RNA-binding proteins (RBPs) but also modify phase transitions of RBPs by recognizing nuclear localization signals (NLSs). Toxic arginine-rich poly-dipeptides from C9orf72 interact with NIRs and cause nucleocytoplasmic transport deficit. However, the molecular basis for the toxicity of arginine-rich poly-dipeptides toward NIRs function as phase modifiers of RBPs remains unidentified. Here we show that arginine-rich poly-dipeptides impede the ability of NIRs to modify phase transitions of RBPs. Isothermal titration calorimetry and size-exclusion chromatography revealed that proline:arginine (PR) poly-dipeptides tightly bind karyopherin-β2 (Kapβ2) at 1:1 ratio. The nuclear magnetic resonances of Kapβ2 perturbed by PR poly-dipeptides partially overlapped with those perturbed by the designed NLS peptide, suggesting that PR poly-dipeptides target the NLS binding site of Kapβ2. The findings offer mechanistic insights into how phase transitions of RBPs are disabled in C9orf72-related neurodegeneration., Nuclear import receptors (NIRs) regulate self-association of RNA-binding proteins as phase modifiers, while C9orf72-derived arginine-rich polydipeptides lead to aberrant phase transitions. Here the authors show in molecular basis how arginine-rich poly-dipeptides impede the ability of NIRs, particularly Kapβ2.

One aspect of the study of the origins of life focuses on how primitive chemistries assembled into the first cells on Earth and how these primitive cells evolved into modern cells. Membraneless droplets generated from liquid-liquid phase separation (LLPS) are one potential primitive cell-like compartment; current research in origins of life includes study of the structure, function, and evolution of such systems. However, the goal of primitive LLPS research is not simply curiosity or striving to understand one of life’s biggest unanswered questions, but also the possibility to discover functions or structures useful for application in the modern day. Many applicational fields, including biotechnology, synthetic biology, and engineering, utilize similar phase-separated structures to accomplish specific functions afforded by LLPS. Here, we briefly review LLPS applied to primitive compartment research and then present some examples of LLPS applied to biomolecule purification, drug delivery, artificial cell construction, waste and pollution management, and flavor encapsulation. Due to a significant focus on similar functions and structures, there appears to be much for origins of life researchers to learn from those working on LLPS in applicational fields, and vice versa, and we hope that such researchers can start meaningful cross-disciplinary collaborations in the future.

Cyanobacteria possess a sophisticated photosynthesis-based metabolism with admirable plasticity. This plasticity is possible via the deep regulation network, the thiol-redox regulations operated by thioredoxin (hereafter, Trx). In this context, we characterized the Trx-m1-deficient mutant strain of Anabaena sp., PCC 7120 (shortly named A.7120), cultivated under nitrogen limitation. Trx-m1 appears to coordinate the nitrogen response and its absence induces large changes in the proteome. Our data clearly indicate that Trx-m1 is crucial for the diazotrophic growth of A.7120. The lack of Trx-m1 resulted in a large differentiation of heterocysts (>20% of total cells), which were barely functional probably due to a weak expression of nitrogenase. In addition, heterocysts of the mutant strain did not display the usual cellular structure of nitrogen-fixative cells. This unveiled why the mutant strain was not able to grow under nitrogen starvation.

ClpB, a bacterial homologue of heat shock protein 104 (Hsp104), can disentangle aggregated proteins with the help of the DnaK, a bacterial Hsp70, and its co-factors. As a member of the expanded superfamily of ATPases associated with diverse cellular activities (AAA(+)), ClpB forms a hexameric ring structure, with each protomer containing two AAA(+) modules, AAA1 and AAA2. A long coiled-coil middle domain (MD) is present in the C-terminal region of the AAA1 and surrounds the main body of the ring. The MD is subdivided into two oppositely directed short coiled-coils, called motif-1 and motif-2. The MD represses the ATPase activity of ClpB, and this repression is reversed by the binding of DnaK to motif-2. To better understand how the MD regulates ClpB activity, here we investigated the roles of motif-1 in ClpB from Thermus thermophilus (TClpB). Using systematic alanine substitution of the conserved charged residues, we identified functionally important residues in motif-1, and using a photoreactive cross-linker and LC-MS/MS analysis, we further explored potential interacting residues. Moreover, we constructed TClpB mutants in which functionally important residues in motif-1 and in other candidate regions were substituted by oppositely charged residues. These analyses revealed that the intra-subunit pair Glu-401–Arg-532 and the inter-subunit pair Asp-404–Arg-180 are functionally important, electrostatically interacting pairs. Considering these structural findings, we conclude that the Glu-401–Arg-532 interaction shifts the equilibrium of the MD conformation to stabilize the activated form and that the Arg-180–Asp-404 interaction contributes to intersubunit signal transduction, essential for ClpB chaperone activities.

While electrophilic reagents for histidine labeling have been developed, we report an umpolung strategy for histidine functionalization. A nucleophilic small molecule, 1-methyl-4-arylurazole, selectively labeled histidine under singlet oxygen (1O2) generation conditions. Rapid histidine labeling can be applied for instant protein labeling. Utilizing the short diffusion distance of 1O2 and a technique to localize the 1O2 generator, a photocatalyst in close proximity to the ligand-binding site, we demonstrated antibody Fc-selective labeling on magnetic beads functionalized with a ruthenium photocatalyst and Fc ligand, ApA. Three histidine residues located around the ApA binding site were identified as labeling sites by liquid chromatography-mass spectrometry analysis. This result suggests that 1O2-mediated histidine labeling can be applied to a proximity labeling reaction on the nanometer scale.

The Mre11-Rad50-Nbs1 complex (MRN) is important for repairing DNA double-strand breaks (DSBs) by homologous recombination (HR). The endonuclease activity of MRN is critical for resecting 5′-ended DNA strands at DSB ends, producing 3′-ended single-strand DNA, a prerequisite for HR. This endonuclease activity is stimulated by Ctp1, the Schizosaccharomyces pombe homolog of human CtIP. Here, with purified proteins, we show that Ctp1 phosphorylation stimulates MRN endonuclease activity by inducing the association of Ctp1 with Nbs1. The highly conserved extreme C terminus of Ctp1 is indispensable for MRN activation. Importantly, a polypeptide composed of the conserved 15 amino acids at the C terminus of Ctp1 (CT15) is sufficient to stimulate Mre11 endonuclease activity. Furthermore, the CT15 equivalent from CtIP can stimulate human MRE11 endonuclease activity, arguing for the generality of this stimulatory mechanism. Thus, we propose that Nbs1-mediated recruitment of CT15 plays a pivotal role in the activation of the Mre11 endonuclease by Ctp1/CtIP.

SummaryContinuous translation elongation, irrespective of amino acid sequences, is a prerequisite for living organisms to produce their proteomes. However, the risk of elongation abortion is concealed within nascent polypeptide products. Negatively charged sequences with occasional intermittent prolines, termed intrinsic ribosome destabilization (IRD) sequences, destabilizes the translating ribosomal complex. Thus, some nascent chain sequences lead to premature translation cessation. Here, we show that the risk of IRD is maximal at the N-terminal regions of proteins encoded by dozens of Escherichia coli genes. In contrast, most potential IRD sequences in the middle of open reading frames remain cryptic. We found two elements in nascent chains that counteract IRD: the nascent polypeptide itself that spans the exit tunnel and its bulky amino acid residues that occupy the tunnel entrance region. Thus, nascent polypeptide products have a built-in ability to ensure elongation continuity by serving as a bridge and thus by protecting the large and small ribosomal subunits from dissociation.

Peroxidase-proximity protein labeling was performed using a hemin-parallel G-quadruplex (G4) complex. A tyrosine labeling reaction using an N-methyl luminol derivative was accelerated in close proximity to the hemin with enhanced peroxidase activity by binding to parallel G4. The TERRA-hemin complex activated the labeling of many RNA-binding proteins, including heterogeneous nuclear ribonucleoproteins, in a HeLa cell lysate.

Herein we report the synthesis and isolation of a shape-persistent Janus protein nanoparticle derived from the biomolecular machine chaperonin GroEL (^AGroEL^B) and its application to DNA-mediated ternary supramolecular copolymerization. To synthesize ^AGroEL^B with two different DNA strands A and B at its opposite apical domains, we utilized the unique biological property of GroEL, i.e., Mg²⁺/ATP-mediated ring exchange between ^AGroEL^A and ^BGroEL^B with their hollow cylindrical double-decker architectures. This exchange event was reported more than 24 years ago but has never been utilized for molecular engineering of GroEL. We leveraged DNA nanotechnology to purely isolate Janus ^AGroEL^B and succeeded in its precision ternary supramolecular copolymerization with two DNA comonomers, A** and B*, that are partially complementary to A and B in ^AGroEL^B, respectively, and programmed to self-dimerize on the other side. Transmission electron microscopy allowed us to confirm the formation of the expected dual-periodic copolymer sequence −(^(B*/B)GroEL^(A/A**/A**/A)GroEL^(B/B*))– in the form of a laterally connected lamellar assembly rather than a single-chain copolymer.

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.

In Escherichia coli, the major bacterial Hsp70 system consists of DnaK, three J-domain proteins (JDPs: DnaJ, CbpA, and DjlA), and one nucleotide exchange factor (NEF: 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 E. coli biofilm formation. Genetic analysis with a complete set of JDP-null mutant strains demonstrated that only DnaJ is essential for survival at high temperature, while DnaJ and CbpA are indispensable in DnaK regulation of curli production. Additionally, we found that DnaJ and CbpA are involved in the expression of the master regulator CsgD through the folding of MlrA; this keeps 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.

We tested whether a short model peptide derived from a group 3 late embryogenesis abundant (G3LEA) protein is able to maintain the fluorescence activity of a red fluorescent protein, mKate2, in the dry state. The fluorescence intensity of mKate2 alone decreased gradually through repeated dehydration-rehydration treatments. However, in the presence of the LEA model peptide, the peak intensity was maintained almost perfectly during such stress treatments, which implies that the three dimensional structure of the active site of mKate2 was protected even under severe desiccation conditions. For comparison, similar experiments were performed with other additives such as a native G3LEA protein, trehalose and BSA, all of whose protective abilities were lower than that of the LEA model peptide., Highlights • We prepared a 22-mer model peptide of a group-3 LEA protein. • The fluorescent peak of a red fluorescent protein was almost lost on drying. • The model peptide suppressed such desiccation-induced damage. • This indicates that the 3D structure of the fluorophore was protected. • The peptide exhibited the highest protective effect among the reagents tested.

Here, we report molecular chaperone GroELs that carry, at their apical domains, multiple DNA strands (ideally 28 DNA strands in total) with defined oligonucleotide (nt) sequences. This design strategy allows for the preparation of GroEL10a and GroEL10b carrying 10-nt DNA strands of 10a and 10b with complementary sequences, respectively, at their apical domains. One-dimensional coassembly of these GroELs is possible to form protein nanotube NT10a/10b with an anomalous thermodynamic stability due to the exceptionally large multivalency for the coassembly. Likewise, comparably stable nanotube NT15c/10d was obtained even when the apical-domain DNA strands (15c and 10d) were partially complementary to one another. Nevertheless, in sharp contrast with NT10a/10b, NT15c/10d, when incubated with DNA 15d, dissociates rapidly and completely because 15d preferentially hybridizes with the DNA strands of 15c in NT15c/10d by displacing those of 10d, to afford a mixture of GroEL15c/15d and GroEL10d. Even in the presence ...

In vitro transcription–translation systems (TX–TL) can synthesize most of individual genes encoded in genomes by using strong promoters and translation initiation sequences. This fact raises a possibility that TX–TL using genome as a template can reconstitute the profile of RNA and proteins in living cells. By using cell extracts and genome prepared from different organisms, here we developed a system for in vitro genome transcription–translation (iGeTT) using bacterial genome and cell extracts, and surveyed de novo synthesis of RNA and proteins. Two-dimensional electrophoresis and nano LC–MS/MS showed that proteins were actually expressed by iGeTT. Quantitation of transcription levels of 50 genes for intracellular homeostasis revealed that the levels of RNA synthesis by iGeTT are highly correlated with those in growth phase cells. Furthermore, activity of iGeTT was influenced by transcription derived from genome structure and gene location in genome. These results suggest that intracellular profiles and characters of genome can be emulated by TX–TL using genome as a template.

Magnetic affinity beads functionalized with lactose and ruthenium/dcbpy complexes were developed. Using MAUra, a catalyst-proximity labelling reagent, the catalytic labeling of lactose-binding proteins was achieved with high selectivity on the beads. The first unbiased identification of cellular endogenous lectins bound to lactose (galectin-1 and galectin-3) was achieved with chemical labelling on the affinity beads.

Protein folding is an essential prerequisite for proteins to execute nearly all cellular functions. There is a growing demand for a simple and robust method to investigate protein folding on a large-scale under the same conditions. We previously developed a global folding assay system, in which proteins translated using an Escherichia coli-based cell-free translation system are centrifuged to quantitate the supernatant fractions. Although the assay is based on the assumption that the supernatants contain the folded native states, the supernatants also include nonnative unstructured proteins. In general, proteases recognize and degrade unstructured proteins, and thus we used a protease to digest the unstructured regions to monitor the folding status. The addition of Lon protease during the translation of proteins unmasked subfractions, not only in the soluble fractions but also in the aggregation-prone fractions. We translated ∼90 E. coli proteins in the protease-inclusion assay, in the absence and presence of chaperones. The folding assay, which sheds light on the molecular mechanisms underlying the aggregate formation and the chaperone effects, can be applied to a large-scale analysis.

The formation of highly condensed, native proteins is important for the development of protein-based drugs and materials. In the cell, various types of liquid droplets with broad functions are formed by the spontaneous condensation of protein, as a physiological response. These droplets lack a surrounding membrane but are phase-separated from the water medium. These types of phase-separated states of proteins have potential applications in biotechnology. Recently, we have developed an artificial phase-separated liquid of condensed native proteins, termed a protein condensate (PC), formed by electrostatic complexation with ionic surfactants. Here we report the applicability of PC formation, studied using an E. coli extract as the protein source. The addition of anionic and cationic surfactants at a specific ratio to the E. coli extract resulted in PC formation. A proteome analysis showed that the PC thus formed contained about 600 kinds of proteins, representing 65% of the uniquely detected proteins and confirming the high versatility of PC formation. A statistical analysis revealed that a variety of types of proteins with a wide range of molecular weights and isoelectric points could form PCs.

Chaperones are essential to maintain the proper folding of various proteins in vivo. The Escherichia coli chaperonin GroEL/GroES (GroE) is one of the best-studied chaperones, and its in vivo substrates have been identified, mainly by mass spectrometry-based proteomic studies. Here, we newly identified 20 in vivo obligate GroE substrates with the aid of data from an in vitro comprehensive analysis. The newly identified substrates have similar physicochemical properties to the known substrates, but their expression levels in vivo were significantly lower. Information from the in vitro comprehensive analysis has the potential to compensate for limitations of the MS-based proteomic approaches.

Thioredoxin (Trx) family proteins perform redox regulation in cells, and they are involved in several other biological processes (e.g. oxidative stress tolerance). In the filamentous cyanobacterium Anabaena sp. PCC7120 (A. 7120), eight Trx isoforms have been identified via genomic analysis. Among these Trx isoforms, the absence of Trx-m1 and TrxC appears to result in oxidative stress in A. 7120 together with alterations of the thylakoid membrane structure and phycobiliprotein composition. To analyze the physiological changes in these Trx disruptants thoroughly, quantitative proteomics was applied. Certainly, the mutants exhibited similar alterations in the proteome including decreased relative abundance of phycobiliproteins and an increased level of proteins involved in amino acid and carbohydrate metabolism. Nevertheless, the results also indicated that the mutants exhibited changes in the relative abundance of different sets of proteins participating in reactive oxygen species detoxification, such as Fe-SOD in Δtrx-m1 and PrxQ in ΔtrxC, suggesting distinct functions of Trx-m1 and TrxC.

Hsp104 solubilizes protein aggregates in cooperation with Hsp70/40. Although the framework of the disaggregase function has been elucidated, the actual process of aggregate solubilization by Hsp104-Hsp70/40 remains poorly understood. Here we developed several methods to investigate the functions of Hsp104 and Hsp70/40 from Saccharomyces cerevisiae, at single-molecule levels. The single-molecule methods, which provide the size distribution of the aggregates, revealed that Hsp70/40 prevented the formation of large aggregates from small aggregates and that the solubilization of the small aggregates required both Hsp104 and Hsp70/40. We directly visualized the individual association-dissociation dynamics of Hsp104 on immobilized aggregates and found that the lifetimes of the Hsp104-aggregate complex are divided into two groups: short (∼4 s) and long (∼30 s). Hsp70/40 stimulated the association of Hsp104 with aggregates and increased the duration of this association. The single-molecule data provide novel insights into the functional mechanism of the Hsp104 disaggregation machine.

The development of nanocarriers that selectively release guest molecules on sensing a particular biological signal is being actively pursued in nanomedicine for diagnostic and therapeutic purposes. Here we report a protein-based nanocarrier that opens in the presence of intracellular adenosine-5'-triphosphate (ATP). The nanocarrier consists of multiple barrel-shaped chaperonin units assembled through coordination with Mg(2+) into a tubular structure that protects guest molecules against biological degradation. When its surface is functionalized with a boronic acid derivative, the nanocarrier is able to enter cells. The hydrolysis of intracellular ATP into adenosine-5'-diphosphate (ADP) induces conformational changes of the chaperonin units, which in turns generate a mechanical force that leads to the disassembly of the tube and release of the guests. This scission occurs with a sigmoidal dependence on ATP concentration, which means that the nanocarrier can differentiate biological environments in terms of the concentration of ATP for selective guest release. Furthermore, biodistribution tests reveal preferential accumulation of the nanocarriers in a tumour tissue.