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Protein folding and disulfide bond formation in theeukaryotic cell Meeting report based on the presentations at the European Network Meeting on Protein Folding and Disulfide Bond Formati

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-vesicle-associated protein-B.

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 interrelafunc-tionships 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

CH1 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 CH1 domain of IgG does not fold properly in isolation The CH1 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 CH1 domain [11,12], and labelled peptide-binding studies are ongoing to reveal the details of the BiP–CH1 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

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, suggestfold-ing 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 funcinforma-tion 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?

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 Cx9C or twin Cx3C 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 vitroas 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 Usinactivat-ing 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

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 Ellg-aard and J R Winther, Department of Biology, University of Copenhagen, Denmark) wish to thank members of their groups for practical help in setting

up the meeting, and D Theodoraki for excellent administrative support The work in the authors’ labo-ratory was supported by grants from the BBSRC (grant number BB⁄ C509582), the Wellcome Trust, the Leverhulme Trust, and the Arthritis Research Campaign

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