meeting report

The life of : the good, the mostly good and the ugly

Richard I Morimoto, Arnold J M Driessen, Ramanujan S Hegde & Thomas Langer The health of the proteome in the face of multiple and diverse challenges directly influences the health of the and the lifespan of the organism. A recent meeting held in Nara, Japan, provided an exciting platform for scientific exchange and provocative discussions on the biology of proteins and homeostasis across multiple scales of analysis and model systems.

The International Conference on Protein Community brought together nearly 300 ­scientists in Japan to exchange ideas on how ­proteins in healthy humans are expressed, folded, translocated, assembled and ­disassembled, and on how such events can go awry, leading to a myriad of protein ­conformational diseases. The meeting, held in Nara, Japan, in September 2010, coincided with the 1,300th birthday of Nara, Japan’s ancient capital, and provided a meditative ­setting for reflecting on the impact of advances in protein community research on biology and Nature America, Inc. All rights reserved. All rights Inc. America, 1 Nature medicine. It also ­provided an opportunity to consider the success of the ­protein ­community © 20 program in Japan since meetings on the stress response (Kyoto, 1989) and on the life of ­proteins (Awaji Island, 2005). The highlights and poster ­presentations. During these socials, and ­accessory factors. Considerable effort has of the Nara meeting were, without question, graduate and postdoctoral students and all of been and continues to be devoted toward the social periods held after long days of talks the speakers sat together on tatami mats at low understanding the ­mechanistic basis of protein tables replete with refreshments and enjoyed maturation and function. each other’s company, while ­participating in Several new concepts and methods are Richard I. Morimoto is in the Department relaxed, spirited and open discussions. ­bringing nascent-chain biology to the of Molecular Biosciences, Northwestern ­forefront of the field. Koreaki Ito (Kyoto University, Evanston, Illinois, USA and at Nascency and nascent-chain biology Sangyo University, Japan), who has ­studied the Rice Institute for Biomedical Research, Replenishment and renewal of the ‘protein ­programmed ribosomal stalling by the Evanston, Illinois, USA. Arnold J. M. Driessen ­community’ of cellular proteins requires Escherichia coli protein SecM, proposed that is in the Department of Molecular Microbiology, new proteins to be synthesized. A theme have heterogeneous ­translation Groningen Biomolecular Sciences and that coursed throughout multiple conference speeds. He presented a two-dimensional gel Biotechnology Institute, Zernike Institute for ­sessions was ‘nascency’, the period between system to examine peptidyl-tRNA species, Advanced Materials, University of Groningen, initiation of the nascent chain and completion ­revealing the nonuniform nature of ­translation. Groningen, The Netherlands. Ramanujan of its assembly into a functioning product. A Ito suggested that during elongation, the S. Hegde is at the National Institute of Child nascent ­protein is in a particularly delicate nascent chain is ‘reviewed’ both inside and Health and Human Development, US National state, prone to ­inappropriate interactions, ­outside the to adjust translation rates, Institutes of Health, Bethesda, Maryland, USA. ­misfolding and aggregation. To avoid these by as-yet-unknown feedback mechanisms. Thomas Langer is at the Institute for Genetics, ­detrimental fates, nascent chains are guided In a global strategy to examine translation, University of Cologne, Cologne, Germany. through their ­maturation by molecular Jonathan Weissman (University of California, e-mail: [email protected] ­chaperones, ­modifying enzymes and ­targeting San Francisco) has developed ribosome

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­profiling, a deep sequencing–based method to lational ­elongation, the nascent polypeptide, the the biogenesis and ­function of pre-protein accurately map the position of ribosomes on ­ribosome and ribosome-associating­ factors. translocases. Toshiya Endo (Nagoya University, all translated mRNAs. Applying this method Upon release from the ribosome, nascent Japan) discussed structural and dynamic aspects to bacterial and mammalian cells, Weissman chains fold with the assistance of chaperones. of protein import into mitochondria. Using an illustrated that sites of translational pausing Presentations by Masasuke Yoshida (Kyoto in vivo cross-linking approach, he showed that were readily detectable, opening the way for Sangyo University) and Hideki Taguchi (Tokyo the ­receptor domain of Tom22 in the genome-wide analyses. By preselecting the Institute of Technology, Yokohama, Japan) outer-membrane TOM40 ­complex changes ribosomes being analyzed, via their association addressed the function and ­mechanism of its ­interaction with another receptor subunit, with trigger factor, Weissman (in collaboration the GroEL . Yoshida used ­various Tom20, to decode the pre-sequence ­targeting with Bernd Bukau) showed it was possible to probes such as antibody accessibility and signal. Tom22 then changes its interaction with monitor precisely when individual nascent ­ digestion to show that a folded the inner-membrane­ pre-sequence ­receptor chains engage this bacterial chaperone. ­substrate is not completely enclosed by GroEL Tim50 to transfer the pre-sequence from In a new example of how non-uniform but rather is transiently accessible to the Tom22 to Tim50. ­translation is used by the cell, Kenji Kohno ­environment. Most substrates spend time in Mechanisms of membrane fusion events (Nara Institute of Science and Technology, a ‘tethered’ state that facilitates folding in the were analyzed in vitro by Hisao Kondo (Kyushu Japan) showed that translational ­pausing GroEL cage by avoiding nonproductive and University, Fukuoka, Japan) and Jodi Nunnari may facilitate mRNA localization. A ­transient kinetically trapped states, from which they (University of California, Davis). Kondo used an pause during Xbp1 translation, ­initiated when can be released to fold rapidly or can escape ER reformation assay to ­identify new ­promoting a short peptide segment enters the ­ribosomal and risk aggregation. Taguchi addressed the and inhibitory factors for ER ­reassembly, and tunnel, provides sufficient time for a ­preceding issue of defining GroEL substrates using a Nunnari described an in vitro mammalian ­hydrophobic segment to ‘drag’ the entire ­combination of in vitro and in vivo assays. mitochondrial fusion assay to address the ­ribosome–mRNA complex to the ­endoplasmic He reinvestigated earlier work by Hartl (Cell ­significance of the two outer-­membrane fusion reticulum (ER) membrane. Xbp1 mRNA is 122, 209–220, 2005) and refined the classes of dynamins, MFN1 and MFN2. MFN1–MFN2 spliced by the ER-resident ­protein Ire1-α substrates that use the GroEL system to avoid trans-complexes showed greater efficacy in when cells ­experience ER stress, ­showing aggregation. He ­suggested that GroEL may fusion as compared to either alone. Moreover, that ­translational pausing influences mRNA help buffer ­destabilizing mutations in ­proteins, MFN2-only complexes also interacted with ­trafficking and splicing. Analogous work from thereby allowing their faster evolution. Bax, the proapoptotic Bcl2 protein, to regulate Ramanujan Hegde (US National Institutes Despite extensive work, little is known about mitochondrial fusion, a result that provides of Health, Bethesda, Maryland) showed that how chaperones such as GroEL or GroES further insight into the unique physiological ­transient pausing, just before ­termination of function to regulate the expression of nascent roles of these mitochondrial fusion proteins. tail-anchored ­membrane proteins, may ­facilitate chains. Takashi Yura (Kyoto University, Japan) Mitochondrial fission, discussed by Katsuyoshi recruitment of ­factors for targeting. One such addressed chaperone-mediated ­feedback Mihara (Kyushu University), is associated with factor, Bat3 (also called Bag6), was suggested ­control in bacteria and described a new the recruitment of the cytoplasmic dynamin- by Hegde to have a dual role in ­targeting and feedback-resistant mutant isolated with a related GTPase Drp1 to mitochondria. Much degradation and to ­function as a triage ­factor transposon inserted upstream of the ftsY gene as in yeast, the mitochondrial outer-membrane that facilitates ­degradation of improperly (encoding the bacterial signal ­recognition protein Fis1 is thought to be a Drp1 receptor in Nature America, Inc. All rights reserved. All rights Inc. America, 1 Nature ­targeted ­membrane proteins. Quite ­satisfyingly, ­particle receptor FtsY). Yura proposed that mammals. However, Mihara showed that the Weissman’s ­ribosomal ­profiling method the SRP-dependent targeting of sigma factor mitochondrial fission and fusion protein Mff, © 20 observed the pauses detected ­biochemically 32 to the bacterial inner membrane ­represents but not Fis1, functions as an essential factor in by Kohno and Hegde. Given the emerging a newly discovered step that facilitates the mitochondrial recruitment of Drp1. evidence for the diverse uses and ­relatively ­inactivation and/or degradation of sigma Although studies on membrane fusion widespread nature of translational pausing, it factor 32, in order to coordinate the cellular events have revealed their importance for seems that nascent-chain processes are tightly responses to changing protein-folding states in ­cellular ­quality control, the dynamic ­behavior regulated and ­choreographed. the ­cytoplasm and membrane. of ­membranes raises questions about how the To further illustrate the ‘dance’ of the nascent ­supply of membrane proteins and ­phospholipids chain, Bernd Bukau (University of Heidelberg, Translocation across membranes is coordinated. Thomas Langer (University of Germany) described how multiple ribosome- Another aspect of the protein community is Cologne, Germany) reported on a conserved associating ­maturation factors—peptide the targeting of proteins from the cytosol to protein network in the ­intermembrane space deformylase (PDF), ­methionine ­aminopeptidase ­organelles, where they have to cross one or of yeast ­mitochondria that determines the (MAP) and ­trigger ­factor—share the crowded more membranes before reaching their final phospholipid ­composition of mitochondrial space ­surrounding the nascent chain. All three ­destination. Mitochondria import about 1,000 membranes. The i-AAA ­protease Yme1, a ­factors bind ­overlapping sites on the ribosome, different proteins from the cytosol. Although ­mitochondrial quality-control enzyme, was but ­different kinetics explains their sequences different machineries for translocation of pre- found to regulate the steady-state ­levels of of action. PDF, for example, has extremely fast proteins have been identified, less is known intrinsically unstable and lipid-specific ­proteins, on- and off-rates and does not discriminate about the regulation of import. Nikolaus Pfanner suggesting that there is ­coordination between between empty and translating ribosomes, (University of Freiburg, Germany) showed mitochondrial protein quality ­control and which allows it to work first. Next, MAP acts on that the mitochondrial proteome contains ­phospholipid ­metabolism. Masato Nakai (Osaka the deformylated nascent chains. Trigger factor, ­numerous protein kinases and ­phosphatases University, Japan) ­discussed the ­composition whose on-rate is very slow, binds only after ~60 and many more ­phosphorylated proteins than of the ­translocase of the inner membrane of residues have been synthesized. It is clear that expected. He discussed how post-translational the chloroplast. This large (1-MDa) ­complex there are tight interrelationships between trans- modification of ­transport components affects with Tic20 as a core component was ­purified,

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­resulting in the ­identification of three ­previously between the ­composition of the machinery for evolution from Ire1 in yeast, worm and flies ­uncharacterized ­components that are all the import into peroxisomes and the ­machinery to ATF6 in mammals. Kazutoshi Mori (Kyoto ­essential for translocation. for ER-associated degradation of proteins. He University) is investigating when and why this In bacteria, protein translocation across discussed mechanistic aspects of the newly switch occurred, using the chicken DT40 cell the cytoplasmic membrane is mediated by identified transient protein-import pore and line and the teleost fish medaka. the ­protein-conducting SecYEG channel an unusual . He also In the ER, oxidoreductases play major roles ­(translocon) and the SecA ATPase. Because ­proposed an export-driven import model both for the ER-associated ­degradation (ERAD) of a wealth of structural information, many for peroxisomal protein import, wherein the of misfolded proteins and for the ­productive of the mechanistic details of the process of ATP- and AAA-peroxin–dependent export folding of newly synthesized ­proteins. Kaz protein translocation, including some that are of the import receptor from the peroxisomal Nagata (Kyoto Sangyo University) examined ­controversial in the field, can now be ­tackled ­membrane to the cytosol is mechanically oxidoreductases by proteomic ­analysis and at the molecular level. Arnold Driessen ­connected to cargo-protein import into the identified several that are oxidized by ERO1-a: (University of Groningen, The Netherlands) organelle. Yukio Fujiki (Kyushu University) ERp46, ERp57, P5 and PDI. Intriguingly, presented the results of single-molecule ­studies addressed various aspects of ­peroxisome ERp72 functions as an inhibitory ­regulator on the oligomeric state of the SecYEG ­complex. homeostasis in mammals. Through a series of the ERO1-a oxidation system without These SecYEG ­complexes were labeled with of biochemical experiments, he showed that ­inhibiting the oxidase activity of PDI. Kenji ­fluorescent markers, ­reconstituted into giant matrix proteins are targeted by the ­cytosolic Inaba (Kyushu University) described the unilamellar vesicles at low protein-to-lipid PTS-receptors, Pex5p and Pex7p, and structure of human Ero1-α in its hyperactive ratios and subjected to fluorescence cross- ­subsequently imported via the Pex5p-docking and inactive forms, revealing how the enzyme ­correlation spectroscopy with single-­molecule complex comprising Pex14p and Pex13p. Pex5p generates protein disulfide bonds de novo sensitivity. This allowed the ­detection of then shuttles back to the cytosol with the aid of in human cells and how its ­activity is tightly SecYEG and the ­determination of pre-protein the AAA ATPase Pex1p–Pex6p complexes on regulated to avoid hyperoxidation of the ER. dynamics in the lipid ­membranes as well as Pex26p and a ­cytosolic factor. Systematic biochemical studies also ­elucidated SecYEG complex formation. Tomoya Tsukazaki the mechanism by which Ero1-α oxidizes PDI (University of Tokyo, Japan) ­presented the The ER: sensing stress and folding specifically and efficiently among nearly 20 X-ray ­crystallographic structure of an ­accessory The ability of the ER to sense and respond to types of PDI-family member ­proteins. David complex of the Sec ­translocase, the SecDF the accumulation of unfolded ­polypeptides Ron (University of Cambridge, UK) reported ­complex, thereby providing new insights about represents another important facet of the on a role for the ER–localized peroxiredoxin how these proteins may ­cooperate with the ­protein community. Peter Walter (University of 4 (PRDX4) in disulfide bond formation. SecYEG and SecA. California, San Francisco) reported that short PRDX4 is redundant with ERO1, ­explaining How transmembrane ­helices are ­recognized peptides bind and ­trigger ­oligomerization of the lack of phenotype in Prdx4-deficient mice. for membrane insertion by the SecYEG the Ire1 ER-lumenal domain in the unfolded However, research on animals harboring ­translocon was addressed by Gunnar von protein response (UPR). He proposed that mutations in ERO1 uncovered PRDX4’s role Heijne (Stockholm University, Sweden), who peptide ligands mimic unfolded proteins in in disulfide bond formation and reveals links described the use of a ­translation-arrest ­peptide the ER lumen, where disordered loops on the between ­oxidative in the ER as an in vivo sensor to map out the forces acting surface of misfolded proteins would ­similarly and ­peroxide production. Nature America, Inc. All rights reserved. All rights Inc. America, 1 Nature on a ­hydrophobic polypeptide ­segment during interact with Ire1, leading to its activation. The importance of ­disulfide bond ­formation its passage through the ribosome–­translocon Oligomerization of the luminal side of the in the folding of many ­secretory proteins © 20 complex. He observed a substantial force ER membrane juxtaposes the Ire1 kinase was highlighted by Hiroshi Kadokura (Nara ­acting on the nascent ­polypeptide chain at the and RNase domains, which, upon ­reaching Institute of Science and Technology), who ­precise point during chain elongation when a threshold concentration, clusters in an detected ­oxidative folding ­intermediates of the ­hydrophobic ­segment is located within the ordered array. Thus, the Ire1 kinase domain substrates and DsbA, a ­bacterial ­disulfide translocon ­channel, a force ­possibly caused by seems to serve as a conformational module bond–­forming enzyme. Analysis of these the ­lateral partitioning of the ­transmembrane that, akin to a rheostat, sets the sensitivity of ­intermediates led him to propose the ­existence segment into the lipid bilayer. Masao Sakaguchi Ire1 ­activation by the degree of unfolding of of ­factors that may affect the ­interaction (University of Hyogo, Japan) ­systematically the protein ligands bound to the kinase ATP- between the enzyme and its substrate to examined the effect of positive charges on binding site. This allows integration of the ­regulate the ­formation of ­disulfide bonds. The ­polypeptide-chain ­movement through the UPR with the metabolic state of the cell. role of the DnaJ-like cochaperones in ER ­quality translocon and showed that they arrest Like many forms of stress, persistent ER stress ­control was ­discussed by Linda Hendershot ­movement at the ­translocon by a ­mechanism can lead to cell death. Feroz Papa (University of (St. Jude Children’s Research Hospital, Memphis, involving electrostatic ­interaction. This California, San Francisco) showed that Ire1-α Tennessee), who showed how ­nonglycosylated ­translocation arrest might be the ­foundation controls a ­commitment switch into ­apoptosis. ER proteins are ­recognized by the for the various topogenic ­functions of positive Under conditions of low ER stress, the kinase machinery. She showed that ­immunoglobulin charges in polypeptides. ­activity of Ire1-α ­autophosphorylates to heavy chains that have the potential to be The mechanistic details of peroxisomal activate XBP1 mRNA splicing and promote secreted are ­associated with ERdj3. In order ­protein import have remained obscure for homeostasis. High-level activation of the to be targeted for ­degradation, ERdj3 must many years, but recent developments have Ire1-α kinase causes ­endonucleolytic decay be released to allow ERdj5 and ERdj4 to bind. unmasked the major players, providing of thousands of mRNAs encoding ER cargos, ERdj4 is ­associated with proteins required further understanding of protein import. such as ­insulin and ­chaperones, and promotes for retrotranslocation to the cytosol, and its Ralf Erdmann (Ruhr-Universität Bochum, apoptosis. Interestingly, the number of such ­overexpression accelerates the ­degradation of Germany) pointed out the striking ­similarity ER ­sensors has changed over the course of non-glycosylated ERAD substrates.

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Stress and aging, misfolding and disease (University of Groningen) described the non- by multiple, dedicated ­chaperones. PAC1 is a Aging, disease and the role of stress response overlapping anti-aggregation activities among chaperone that assists the initial assembly step pathways that upregulate chaperones have been the gene families encoding the large ­chaperones. of 20S , the ­catalytic core of 26S topics of considerable interest to the protein Focusing on the diverse DNA-J family of ­proteasomes, and is required for ­embryonic community. Whereas robust ­quality control cochaperones, Kampinga showed that DNAJB6 development and ­neuronal ­proliferation after pathways and stress responses act to detect, and DNAJB8 are the most ­efficient suppressors birth. PAC1-deficient ­hepatocytes were shown destroy and mitigate the effects of ­misfolded of ­aggregation and ­toxicity of ­polyglutamine to contain normal ­levels of 26S ­proteasomes proteins, an all-too-common ­outcome is proteins. He further showed that their ­activity is but deficient levels of free latent 20S their aggregation. Although almost all work ­regulated by ­histone deacetylases and retained ­proteasomes, ­leading to ­inefficient ­degradation to this point has focused on cell-autonomous in cells with an abrogated HSR. This implies that of ­ubiquitinated ­proteins. stress responses in single ­eukaryotic cells, the members of the DNA-J family of ­chaperones Other quality control systems, such as ­diseases of protein misfolding occur in highly could prevent disease, even in aged individuals or organellar quality control, ­interconnected multicellular organisms. whose HSR has declined. involve intense membrane dynamics, whose Presentations by Rick Morimoto The misfolding theme was addressed further ­mechanisms and regulation were discussed in (Northwestern University, Evanston, Illinois) by Chris Dobson (University of Cambridge), several talks. Yoshinori Ohsumi (Tokyo Institute and Andrew Dillin (Salk Institute for Biological who provided an overview of ­misfolding of Technology, Japan) provided ­evidence for an Studies, La Jolla, California) stressed the and ­aggregation, pointing out that a protein’s Atg9-containing ­intermediate structure in the ­importance of ­complementing cell culture native state is in fact an unusual and ­special formation of yeast ­—the most ­studies with ­analyses of ­animals. For the ­situation dominated by highly specific side critical event in autophagy—that results in the ­cytosolic heat-shock response (HSR), Morimoto chain ­interactions that force the main chain sequestration of damaged material. Mitophagy, showed that two thermosensory neurons in the into a ­specific conformation. By contrast, the an evolutionarily conserved ­degradation process ­nematode Caenorhabditis ­elegans can ­regulate β-sheet–rich amyloid structure is a generic selective for mitochondria, involves autophagy- the HSR in the worm’s somatic ­tissues. An RNA ­protein ­structure dominated by main chain related proteins essential for ­ ­interference screen was used to ­identify many interactions, largely irrespective of the side formation. Specificity is ensured by the of the signaling pathways involved, ­allowing chains. This makes ­amyloid ­formation, actions of transmembrane ­receptor proteins. the importance of the non-­autonomous HSR ­associated with ­degenerative diseases, a The mitophagy-specific receptor Atg32 in to be examined. Remarkably, by ­permitting ­possible ­outcome for many, if not most, ­cellular ­budding yeast recruits Atg8 and Atg11 to the the ­autonomous HSR to operate, the worms ­proteins. Furthermore, the ­amyloid state can surface of ­mitochondria. Koji Okamoto (Osaka improved their protein ­homeostasis and be propagated by a nucleated ­seeding process, University) showed that peroxisome-anchored ­better dealt with ­misfolded proteins, but at the which Keiichi Higuchi (Shinshu University, Atg32 can ­promote pexophagy, suggesting ­substantial cost of reduced fecundity. Also using Matsumoto, Japan) suggested could underlie that this ­protein receptor contains modules C. ­elegans, Dillin showed that the ­mitochondrial transmission of certain amyloidoses among ­compatible with autophagy-mediated organelle unfolded protein response (mtUPR) can act ­animals such as cheetahs and mice, to cause ­degradation. Further, Okamoto reported on the non-­autonomously in the cell by means of ­disease. This view is ­consistent with work by phosphorylation of Atg32, which is critical for ­hypothesized ­‘mitokines’. Remarkably, ­induction Hideki Nishitoh (University of Tokyo), who mitophagy, and showed that phosphorylation of the mtUPR in ­neurons can ­activate it in other examined the toxic function of 130 ­different depends on Atg1, a protein kinase required in Nature America, Inc. All rights reserved. All rights Inc. America, 1 Nature ­tissues such as the ­intestine, with downstream amyotrophic lateral sclerosis (ALS)-linked all autophagy-related pathways. consequences for electron ­transport chain SOD1 mutations. These SOD1 mutants © 20 ­function and, ­ultimately, longevity. These ­trigger ER stress through the ­interaction Prospects for the protein community ­studies highlighted the ­complexities of and with Derlin-1, a component of the ERAD The 2010 Protein Community Meeting was ­differences in stress response regulation in cells ­machinery, ­eventually leading to ER stress– a success on multiple levels. Over half of the that are isolated in the laboratory versus those mediated motor neuron death. ­participants were graduate or ­postdoctoral found in situ. Clearance of damaged proteins is an ­essential ­students, who contributed tremendous energy Ulrich Hartl (Max Planck Institute of component of the protein ­community and and ideas to the discussions, pointing to a , Martinsried, Germany) described was represented by talks on the long and promising future for the field. As how appropriate chaperone ­levels can maintain and on autophagy. Kunitoshi Yamanaka we ­understand more about the ­fundamental protein homeostasis and help the cell avoid toxic (Kumamoto University, Japan) described ­biology of proteins and how proteins can exist aggregates. Why ­amyloid should be detrimental how the ­-selective AAA (ATPases in ­multiple functional states that affect the remains poorly ­understood, so to address this ­associated with diverse cellular activities) health of the cell, we increasingly appreciate question, Hartl used synthetic β-sheet–forming ­chaperone p97 (also known as CDC48) plays that ­protein dysregulation contributes to the proteins that generate ­artificial amyloids to an ­important role in sex determination in complexity of human disease and may be a core identify the beta-interactome­ by proteomic C. ­elegans by ­regulating CUL-2–mediated problem of aging and age-associated disease. analyses. The ­identification of ­cellular proteins TRA-1A ­degradation by ­proteasomes. Keiji The prospects for protein community research that ­coaggregate with amyloids suggests that Tanaka (Tokyo Metropolitan Institute of are rich with opportunity. aggregation interferes with the basic defense Medical Science, Japan) then explained how mechanisms of the cell. Continuing the theme the ­proteasome, which has a highly complex COMPETING FINANCIAL INTERESTS of protein aggregation, Harm Kampinga ­structure, is assembled with high precision The authors declare no competing financial interests.

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