meeting report
The life of proteins: 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 cell 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 protein 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 chaperone 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 ribosomes 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 ribosome 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 cytosol 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 chaperonin. 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 protease 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 deubiquitinating enzyme. 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 protein folding 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 Hsp70 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 proteasomes, 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 autophagy 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 autophagosomes—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 autophagosome 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 Biochemistry, Martinsried, Germany) described was represented by talks on the proteasome 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 ubiquitin-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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