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

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-

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

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

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

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-

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

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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