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MEETING REPORT Protein folding and disulfide bond formation in the eukaryotic cell Meeting report based on the presentations at the European Network Meeting on Protein Folding and Disulfide Bond Formation 2009 (Elsinore, Denmark) Adam M. Benham Biological and Biomedical Sciences, Durham University, UK Introduction Protein folding in a living cell does not usually happen spontaneously. Many factors, including chaperones, regulatory enzymes, and redox components, exist to help different proteins find the right path to their shape and activity [1]. Protein misfolding can lead to disease through either loss or gain of an individual protein’s function [2,3], or through more general mechanisms, e.g. when mischarged tRNAs result in misfolded protein accumulation in the neuronal cytoplasm [4]. The intracellular environment in which protein folding occurs has a major influence on how a protein folds [5]. For example, the folding status of nucleoporins has emerged as an important regulatory step in governing the transport of proteins through the nuclear pore complex [6]. Regulation of protein quality control is also important at the surface or outside the cell, one example being to control fibrin activity during blood coagulation [7]. However, the meeting in Keywords chaperone; endoplasmic reticulum; mitochondria; protein disulfide isomerase; protein folding; redox regulation Correspondence A. Benham, Biological and Biomedical Sciences, Durham University, South Road, Durham DH1 3LE, UK Fax: +44 191 334 1201 Tel: +44 191 334 1259 E-mail: Adam.Benham@durham.ac.uk (Received 19 August 2009, revised 23 September 2009, accepted 25 September 2009) doi:10.1111/j.1742-4658.2009.07409.x The endoplasmic reticulum (ER) plays a critical role as a compartment for protein folding in eukaryotic cells. Defects in protein folding contribute to a growing list of diseases, and advances in our understanding of the molec- ular details of protein folding are helping to provide more efficient ways of producing recombinant proteins for industrial and medicinal use. More- over, research performed in recent years has shown the importance of the ER as a signalling compartment that contributes to overall cellular homeo- stasis. Hamlet’s castle provided a stunning backdrop for the latest Euro- pean network meeting to discuss this subject matter in Elsinore, Denmark, from 3 to 5 June 2009. Organized by researchers at the Department of Biology, University of Copenhagen, the meeting featured 20 talks by both established names and younger scientists, focusing on topics such as oxida- tive protein folding and maturation (in particular in the ER, but also in other compartments), cellular redox regulation, ER-associated degradation, and the unfolded protein response. Exciting new advances were presented, and the intimate setting with about 50 participants provided an excellent opportunity to discuss current key questions in the field. Abbreviations ER, endoplasmic reticulum; Ero1, endoplasmic reticulum oxidoreductase 1; HLA, human leukocyte antigen; LDLR, low-density lipoprotein receptor; MHC, major histocompatibility complex; PDI, protein disulfide isomerase; UPR, unfolded protein response; VAP-B, vesicle- associated membrane protein-associated protein-B. FEBS Journal 276 (2009) 6905–6911 ª 2009 The Author Journal compilation ª 2009 FEBS 6905 Elsinore focused mainly on protein folding in the eukaryotic endoplasmic reticulum (ER) and mitochon- dria. An important consideration in these compart- ments is the requirement for disulfide bonds, which form between the SH groups (thiols) of two cysteine residues in a relatively oxidizing environment. The disulfide bond offers structural stability, and may con- tribute to a native protein’s enzymatic function or reg- ulation. The formation of protein disulfides is catalysed by thiol–disulfide oxidoreductases through thiol–disulfide exchange reactions. These enzymes have the capacity to oxidize, reduce or isomerize disulfide bonds, and how and when these different activities come into play is the subject of much current research. There are also a surprisingly large number of thiol– disulfide oxidoreductase and related genes in the human genome, and understanding their specific func- tions and interrelationships is of considerable impor- tance, given their value to industry and medicine. Protein folding in vitro and in vivo In vitro approaches have long provided the basis on which to understand the complexity of protein folding in vivo. S. Ventura (Barcelona, Spain) discussed the challenges of elucidating the folding pathways of disul- fide bond-containing proteins [8]. The Ventura group and others have made considerable progress in studying three model proteins from human pests, namely the car- boxypeptidase inhibitors from the leech and from the tick, and the leech tryptase inhibitor. Structures of these intermediates have been solved, and the folding path- ways reveal some surprises. The tick carboxypeptidase inhibitor turns out to be rather flexible, and can fold either from the N-terminal domain first or the C-termi- nal domain first. The restrictions imposed by the fold- ing pathway limit the theoretical possibilities for forming disulfide bonds. Lessons learned from these model proteins are now being applied to bigger chal- lenges, such as the low-density lipoprotein receptor (LDLR) and its domains [9]. Calcium seems to compete with disulfide bond formation in the LDLR, and in the presence of this cation, non-native disulfide bonds predominate. LDLR mutants that cause familial hyper- cholesterolaemia tend to end up in insoluble aggregates because they cannot attain the native state. Folding of immunoglobulin domain proteins Antibodies have long been favourite subjects for stud- ies on protein folding. They are of crucial biological importance for adaptive immune defence, and can cause serious mischief when they are inappropriately produced, e.g. in autoimmune diseases. Improvements in the efficiency of antibody production in heterolo- gous systems would also be welcomed by industry. M. Feige and M. Marcinowski from the Buchner labora- tory (Munich, Germany) discussed some very elegant work showing that the soft spot of an antibody is its C H 1 domain [10]. Antibodies are composed of a basic unit of two heavy and two light chains, each of which has repeating units of ‘constant’ or ‘variable’ immuno- globulin domains, with the variable regions contribut- ing to antibody diversity. NMR analysis showed that, unlike the CL domain, the C H 1 domain of IgG does not fold properly in isolation. The C H 1 domain needs the context of CL for productive antibody folding and assembly, and the mechanism appears to be conserved between different immunoglobulin classes and between species. The data suggest that proline isomerization at the conserved Pro32 is a rate-limiting step that pre- cedes covalent linkage of the heavy and light chains. This is consistent with reports that the ER chaperone BiP targets the C H 1 domain [11,12], and labelled peptide-binding studies are ongoing to reveal the details of the BiP–C H 1 interaction. For some antibodies, assembly into a light chain– heavy chain complex is not the end of the story. M. Cortini from the Sitia group (Milan, Italy) explained how IgM has the added problem of forming pentamers in the ER prior to export [13]. It seems that IgM uti- lizes a platform consisting of the ER–Golgi intermedi- ate compartment transport protein 53 (ERGIC53) and the protein disulfide isomerase (PDI) family member ERp44. IgM glycosylation mutants have been used to investigate how the assembly process occurs, and it appears that glycans may act as molecular spacers to ensure correct positioning of IgM subunits during pen- tamerization. Major histocompatibility complex (MHC) molecules are also critical to the immune response, and share structural similarities with anti- bodies, being built from the same domain prototype. M. van Lith (Durham, UK) described how quality control of classical MHC class II proteins compared with that of the MHC chaperone human leukocyte antigen (HLA)-DM. HLA-DM does not bind antigenic peptides, unlike its cousins HLA-DP, HLA-DQ and HLA-DR [14]. Interestingly, the DMa glycan at Asn165 is partially endoglycosidase H-sensitive, pro- viding another example of how topological constraints can influence post-translational modifications. It is not just endogenous proteins that fold in the ER. Infectious viruses rely on the host ER to help manufacture their coat proteins, and A. Land (Utr- echt, The Netherlands) described how complex this is Protein folding and disulfide formation A. M. Benham 6906 FEBS Journal 276 (2009) 6905–6911 ª 2009 The Author Journal compilation ª 2009 FEBS for HIV. The major envelope protein of HIV is gp160, which is cleaved into gp120 and gp41 subunits. Gp160 has 10 disulfide bonds and folds very slowly in the ER, but needs to do so in order to keep the amount of productively folded protein high. The leader peptide of gp160 is removed after synthesis, and its rate of removal both determines, and is determined by, fold- ing of the protein, suggesting that the signal peptide helps to control the efficiency of envelope production [15]. PDI PDI is the father of disulfide bond catalysts, but has long resisted attempts to solve its crystal structure [16]. Now, PDI has yielded its secrets, and H. Schindelin (Wu ¨ rzburg, Germany) described how this protein’s form was finally revealed [17,18]. Two yeast PDI struc- tures, a ‘twisted U’ and an ‘open boat’, are related by large-scale conformational changes, and provide snap- shots of how the protein might bind substrates and interact with electron acceptors such as ER oxidore- ductase 1 (Ero1). Interpreting the crystal structures of PDI would not have been possible without the knowl- edge gained from a series of NMR studies on the protein, and K. Wallis from R. Freedman’s laboratory (Warwick, UK) explained the latest approaches to studying ligand binding to human PDI in solution. The x-linker region, a 20 amino acid spacer between the b¢ and a¢ domains, is likely to play an important role in controlling binding of substrates [19,20]. Look- ing forward, the combination of dynamic NMR stud- ies and specific higher-resolution crystal structures and costructures will surely provide a wealth of informa- tion about the function of PDI and its homologues over the next few years. Redox control in the ER The PDI–Ero1 system of catalysing disulfide bonds in the ER has received a lot of attention recently, but M. Csala (Budapest, Hungary) reminded us that proteins and glutathione are not the only redox-active compo- nents of the ER. Pyridine nucleotides (NADP + ⁄ NADPH) have been rather overlooked, but may be particularly important in some diseases, such as meta- bolic syndrome, and during cortisol generation in adi- pogenesis [21]. L. Ruddock (Oulu, Finland) reinforced the point that alternative pathways for disulfide bond formation in the ER should not be ignored: glutathi- one, vitamin K and ascorbate all have a role to play in determining the functional ER redox environment. Consideration should be given to whether significant peroxide generation occurs during disulfide bond for- mation, an issue that has implications for cellular stress and hypoxia [22]. The ER may well harbour PDI peroxidases that minimize exposure to intracellu- lar peroxide and reduce the risk of free radical gene- ration. Indeed, the ER shows great resilience to oxidative insults. C. Appenzeller-Herzog from the Ellgaard group (Copenhagen, Denmark; Basel, Switzerland) presented data showing how cells exposed to reducing stress rapidly re-establish equilibrium, a process that may be controlled, in part, by Ero1a [23]. In this connection, peroxiredoxin IV is emerging as a key player in dealing with the consequences of disul- fide bond formation in the ER [24]. N. Bullied (Man- chester, UK) described how this protein was identified in a client screen using the PDI homolog ERp46 as bait. Peroxiredoxin IV knockdown results in hypersen- sitivity to ER stress, and it can sense the reduction potential of the ER by cycling between the cysteine thiol form and the hyperoxidized cysteine sulfenic acid (–SOH) form. The molecular details of how this enzyme operates alongside oxidative and reductive pathways of protein folding, and thus functions to help maintain balanced ER redox conditions, are sure to emerge in the coming years. Protein targeting and disulfide bond formation in mitochondria Not only must proteins fold in the ER, but they must also be targeted to the right intracellular or extracel- lular destination. The problem is even more challeng- ing for C-tail-anchored proteins, which must be targeted to membranes post-translationally [25]. Vesi- cle-associated membrane protein-associated protein-B (VAP-B) has a role in vesicle trafficking, and the mutation P56S in VAP-B can cause the familial neu- rodegenerative disorder amyotrophic lateral sclerosis type 8. However, the underlying mechanism of this tail-anchored protein’s role in disease is not estab- lished. E. Fasana from the Borgese group (Milan, Italy) described some very interesting data, showing that the transmembrane segment of VAP-B is rather inefficient at inserting into membranes. Mutant VAP-B does not route properly, but accumulates in ER membrane-derived inclusions, which contain a number of ER chaperones, such as calnexin and PDI. As VAP-B is ubiquitously expressed, it will be revealing to determine why the defect hits neurons so hard. Is loss of function of vesicle trafficking over long dis- tances, or gain of ER toxicity in long-lived cells, at the root of the disease process in amyotrophic lateral sclerosis type 8? A. M. Benham Protein folding and disulfide formation FEBS Journal 276 (2009) 6905–6911 ª 2009 The Author Journal compilation ª 2009 FEBS 6907 The ER is not the only compartment where disulfide bonds are made. The intermembrane space of the mitochondrion supports similar activity [26]. J. Riemer (Kaiserslautern, Germany) and B. Morgan from H. Lu’s group (Manchester, UK) described how Mia40 and Erv1 catalyse disulfide bond formation, with the resultant transfer of electrons to cytochrome c, gener- ating water from oxygen through the action of cyto- chrome c oxidase. This formation of disulfide bonds drives substrate import of some intermembrane space proteins from the cytosol through the translocator of the outer mitochondrial membrane complex. The clas- sic substrates of the Erv1–Mia40 pathway are small proteins with either twin Cx 9 C or twin Cx 3 C motifs, e.g. Cox19 and Tim9. The oxidative folding of Tim9 determines its rate of transport, and zinc may play a chaperone-like role, by initiating a conformational change of Tim9 prior to transport [27]. Recombinant proteins and toxins Sometimes, thiol–disulfide chemistry can surprise us in vitro as well as in vivo. R. Nielsen from the Winther laboratory (Copenhagen and NovoNordisk, Denmark) introduced the concept of protein trisulfides, and explained how these unexpected covalent modifications could occur during industrial recombinant protein pro- duction of growth hormone, interleukin-6, and super- oxide dismutase [28]. Although this modification probably occurs during the workup of proteins, a bio- logical effect of this conversion should not necessarily be excluded. The proposed mechanism of generation of a trisulfide from a disulfide involves the production of hydrogen sulfide, which has been ascribed a poten- tial signalling role [29]. Protein folding in plants probably receives much less attention than it deserves. Plants produce some potent toxins, such as the castor bean heterodimer ricin, which is fatal to humans at a 500 lg dose. R. Marshall and R. Spooner (Warwick, UK) presented work that explained how ricin synthesis is controlled, and how it exerts its toxic effect on mammalian cells by inactivat- ing the ribosome. Using tobacco protoplasts to study individual ricin chains, it is possible to dissect out the folding and trafficking requirements that take the pro- tein into the vacuole for storage, or into the cytosol from the ER. The AAA-ATPase p97 plays a critical role in this decision process [30]. Upon binding to the surface of mammalian cells, ricin gains access to the cell’s interior by retrograde transport. The ricin A chain is retrotranslocated into the cytosol from the ER, but first has to be reduced, making ricin A an excellent model for studying the molecular details of ER-associated degradation. Current work is focused on the Hsp40–Hsc70 system, which may control the decision between ricin A retention in the ER and its ubiquitination, and hence proteasomal degradation, in the cytoplasm [31]. Protein turnover Oxidative protein folding in the cytoplasm itself is rare. Instead, the reductive ‘antioxidant’ environment is (partly) maintained by the oxidoreductase thiore- doxin. R. Hartmann-Petersen (Copenhagen, Denmark) discussed a thioredoxin-related protein called Txn11 ⁄ TRP32, which has emerged as a new subunit of the 26S proteasome [32,32a]. The expression of Txn11 is widespread, but its specific function in proteasomal activity is not yet known. However, there are some tantalizing clues to its job: the Txn11 subunit targets eEF1A, which, besides its role in protein translation, has been reported to mediate proteasomal degradation of misfolded proteins. Alternatively, Txn11 may play a role in proteasome assembly. Proteasomal destruction of proteins is an energy- consuming business, and the cell tries hard to fold miscreant proteins before giving up on them. The unfolded protein response (UPR) is one such mecha- nism, whereby membrane-spanning stress sensors such as Ire1 detect unfolded proteins in the ER and facili- tate the upregulation of ER chaperones. E. van Anken, from P. Walter’s laboratory (San Francisco, CA, USA), described how yeast has been used as a model to visualize Ire1p ER stress signalling foci and to track their appearance microscopically in real time [33]. This major development in imaging the ER stress response in vivo promises to provide a wealth of data about the physiological management of ER stress, and it will be interesting to see how similar technologies can be used to study stress foci in higher organisms, where the ER stress response is more complex [33a]. Conclusions There have been a number of major advances in the field over the past few years. One of the most impor- tant breakthroughs has been the emergence of the Ero1 [34] and PDI crystal structures [17]. This has been accompanied by a wave of related PDI family structures [35–37], and raises the real prospect that PDI–Ero1 or PDI–client structures may be solved in the near future. Such cocrystals will help to provide a molecular explanation of how a disulfide isomer- ase ⁄ oxidoreductase solves the problem of recognizing non-native structural elements in specific proteins. A Protein folding and disulfide formation A. M. Benham 6908 FEBS Journal 276 (2009) 6905–6911 ª 2009 The Author Journal compilation ª 2009 FEBS second important advance in this area is the emergence of other redox regulatory mechanisms in the ER. Given the intense interest in oxidative stress and dis- ease, this area will probably provide a focus for much research activity and discovery in the next few years. The realization that mitochondria also host a disulfide bond relay system spawned a flurry of activity in this area, and the race is still on to define the full range of clients, their effects on mitochondrial metabolism, and how misfolded mitochondrial proteins are eliminated. Another key concept is that folding quickly is not always the best thing for a protein, as demonstrated at this meeting for HIV gp160 [15]. One would expect to see more examples of how folding speed affects a pro- tein’s biological activity emerge in the literature over the next few years. The detection and turnover of mis- folded ER proteins (by the UPR–ER-associated degra- dation–proteasome axis) has been a hot topic for some time, and we are likely to continue to witness major breakthroughs here as well, particularly with the advent of tools for the visualization of UPR foci in living cells. Pharmaceuticals that target, or behave as, molecular chaperones have been developed, e.g. in an animal model of type 2 diabetes [38]. The tailoring of such compounds to steer the folding and turnover of specific target proteins in the clinic should therefore be possible for a number of diseases where protein misfolding is a major component [39]. In a round-up discussion led by I. Braakman (Utr- echt, The Netherlands), the community identified a number of key questions that still need to be addressed to take this exciting area of research forwards into the future. Indeed, obtaining accurate measurements of the concentrations, distributions and flux of all the main players involved in protein folding in the ER in vivo is still a fundamental challenge. Whereas we know what many protein foldases and catalysts can do, we do not always know what they actually do in their tissue-spe- cific environments. Oxygen and other gases, redox and nutrient conditions are likely to differ considerably both within and between tissues, and are not always the same as the controlled conditions used to study cells and proteins in tissue culture or in vitro. The meeting concluded with real enthusiasm for the chal- lenges ahead, and a sense of pride that the field was moving forwards so rapidly, with plenty of room for cooperation, collaboration and open discussion of new scientific concepts. Acknowledgements The author thanks the participants of the meeting for critical comments on the manuscript, and for sharing details of unpublished work. The organizers (L. 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MEETING REPORT Protein folding and disulfide bond formation in the eukaryotic cell Meeting report based on the presentations at the European Network Meeting on Protein Folding and Disulfide Bond Formation. in the human genome, and understanding their specific func- tions and interrelationships is of considerable impor- tance, given their value to industry and medicine. Protein folding in vitro and. reductive pathways of protein folding, and thus functions to help maintain balanced ER redox conditions, are sure to emerge in the coming years. Protein targeting and disulfide bond formation in mitochondria Not