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Protein folding: mechanisms and role in disease
(Ulrich Hartl)
At a glance:
Protein molecules are responsible for almost all biological functions in cells. In order to fulfil
their various biological roles, these chain-like molecules must fold into precise three-dimensional
shapes. Incorrect folding and clumping together of proteins is being recognized as the cause for a
growing number of age-related diseases, including Alzheimer’s and Parkinson’s disease as well
as other neurodegenerative disorders.
Definition of topic:
Most biological functions in living cells are performed by proteins, chain-polymers of amino
acids that are synthesized on ribosomes based on genetic information. Upon synthesis, protein
chains must fold into unique three-dimensional structures in order to become biologically active.
While in the test-tube this folding process can occur spontaneously, in the cell most proteins
require assistance for proper folding by so-called ‘molecular chaperones’. These are specialized
proteins which protect other, not-yet folded proteins from misfolding and clumping together
(aggregating) in the highly crowded cellular environment. However, proteins do not always fold
correctly, despite the existence of a complex cellular machinery of protein quality control. In
particular, an increasing number of neurodegenerative diseases have been recognized in recent
years to be caused by the accumulation of protein aggregates in the brain and other parts of the
central nervous system. These disorders, including Alzheimer’s dementia, Parkinson’s disease
and Chorea Huntington, are typically age-related and impose a large social and economic burden
on the aging societies of industrialized nations. Currently, there is no cure for any of these
diseases, and it is believed that a concerted research effort in the areas of protein folding,
combined with a systems biology analysis of the networks of protein quality control will provide
the knowledge base for the development of new therapeutic strategies.
Status of the field
Human cells contain thousands of different proteins that fulfill a large variety of essential
functions in metabolism, cell regulation and development. The protein make-up of a cell
constitutes its ‘proteome’. How proteins are produced and reach their functional forms has long
been one of the most fascinating problems in the life sciences. Each protein is characterized by a
specific sequence of amino acids, small building blocks that are joined together like beads on a
string by the ribosomes in a process called translation. There are 20 different amino acids and
their sequence in a specific protein is determined by the DNA information in the corresponding
gene. To produce a protein the ribosome reads a copy of the respective DNA, the so-called
messenger RNA and translates it into the amino acid sequence it encodes. However, a newlymade or ‘nascent’ protein chain is biologically quite useless. In order to become functionally
active, the chain must adopt a precisely defined three-dimensional pattern, its ‘fold’ (Figure 1A).
The information that specifies the fold is contained in the amino acid sequence, and thus in the
corresponding gene, but how it is successfully realized remains one of the most fundamental
problems in biology, despite more than 50 years of research. Part of the problem is that the
number of different shapes a protein chain can fold into is astronomically large and it is not yet
entirely clear how proteins manage to find the single biologically active conformation out of the
1030 or so possible states 1. Proteins are on average 100 to 500 amino acids in length. To reduce
the magnitude of the folding problem, larger proteins are divided into units (domains) that fold
separately. The folding process is thermodynamically driven by the hydrophobic effect, basically
the tendency of the water-rejecting (hydrophobic) amino acids to interact with one another and
form a hydrophobic core while the water-loving (hydrophilic) amino acids remain at the surface.
As a result, the expanded protein chain rapidly collapses into a globular structure. This drastically
reduces the available conformational space (Figure 1B). Rearrangement steps within the globule
finally give rise to the correct amino acid packing that usually corresponds to the most stable,
biologically active state. Small proteins can complete this task within milliseconds, whereas large
ones may take seconds to many minutes.
Given the complexity of the folding process, it is not too surprising that things can go wrong. The
main problem is that not-yet folded proteins are sticky, because their hydrophobic amino acids
are not yet buried completely. As a result, they may clump together into intractable aggregates
(Figure 2), not unlike the transition egg white undergoes when heated. The tendency to aggregate
is strongly enhanced due to the ‘crowding’ of the cellular environment – the fact that the cellular
solution is highly concentrated in proteins and other large molecules reaching concentration up to
400 gram per liter. How then are proteins able to fold at all? The solution was provided by the
discovery of ‘molecular chaperones’ in the late 1980s that led to a paradigm shift in our
understanding of how proteins fold in the cell. These components, themselves proteins, help other
protein chains to fold efficiently, mostly by preventing aggregation, similar to the human
chaperone that prevents unwanted interactions between people. The molecular chaperones
achieve this by shielding the sticky hydrophobic surfaces of unfolded or misfolded protein
molecules. Regulated cycles of chaperone binding and release then result in suppression of
aggregation and productive folding 2 (Figure 2). One particularly fascinating class of molecular
chaperones, called ‘chaperonins’, form large cylindrical complexes with a central cavity and a lid.
They are present in all types of cell and function by encapsulating an unfolded protein chain in a
cage-like structure, allowing it to fold in isolation, unimpaired by aggregation (Figure 3). The lid
of the folding chamber opens after 10 seconds or so to discharge folded protein.
Numerous classes of chaperones exist and many of them are so-called stress proteins or heatshock proteins, i.e. they are increasingly synthesized by cells under conditions of stress, in which
proteins may unfold and give rise to aggregation. It is now known that these factors function as
part of a complex network of protein quality control 3. This system also includes machinery for
protein degradation such as the proteasome, another cylindrical complex that shredders terminally
misfolded proteins into small fragments which are further cleaved into amino acids for re-use in
the synthesis of new proteins.
Why is protein misfolding and aggregation such an important problem and why have cells
evolved such an elobarate machinery to prevent it? The reasons are two-fold: When a protein fails
to fold correctly upon synthesis or misfolds at a later stage in its cellular life time, it can no longer
fulfill its biological function. This may happen as a result of inherited mutations in proteins,
which cause a change in the amino acid sequence. Perhaps the most famous example is the
disease cystic fibrosis, which in most patients is caused by the deletion of a single amino acid in
the protein chain of cystic fibrosis conductance regulator (CFTR), a transporter for chloride ions.
Another important example concerns the tumor suppressor protein p53, which when mutated
loses stability and consequently its ability to block uncontrolled cell growth, thereby giving rise
to cancer. However, the second, and probably even more critical danger arising from aberrant
folding lies in the fact that misfolded and aggregated proteins are toxic to cells, apparently
because their accumulation interferes with a variety of cell functions. This insight is relatively
new and provides the pathomechanistic basis for many age-related diseases of the nervous
system, such as Alzheimer’s and Parkinson’s disease, Chorea Huntington and the prion diseases
4,5
(Figure 4A). In all these neurodegenerative disorders certain disease proteins accumulate as
misfolded and aggregated species in the brain, either inside or around neuronal cells (Figure 4B).
In Alzheimer’s disease, for example, a small protein fragment called Aβ accumulates initially in
the hippocampus, disturbing the complex neural networks of this brain region, resulting in cell
death and loss of memory function (Figure 4A). It turns out that a particular form of protein
aggregate is most intimately involved with these toxic phenomena. These aggregates are highly
ordered fibrils that are known as amyloid (Figure 5). They are generated from smaller, less
ordered protein clumps, so-called soluble oligomers, which many believe are the most toxic
misfolded protein species.
Why are these diseases mostly afflicting the elderly? Strikingly, recent work in model organisms
such as the worm C. elegans suggests that the capacity of protein quality control declines during
aging. In other words, as we age, our body gradually loses the ability to prevent the accumulation
of misfolded proteins. At some point this may result in a catastrophic breakdown of protein
homeostasis, and in the manifestation of disease 6.
Key scientific questions and opportunities
The emerging consensus that protein misfolding is the cause of a number of age-related diseases
now offers the opportunity to develop a generic therapy for a group of devastating ailments that
are increasingly recognized as a major challenge to the health systems of industrialized nations.
Research in the field of protein folding, formerly merely of academic interest, has led to
surprising insights into biology that are of enormous medical significance. These opportunities
are recognized internationally, as reflected by major research initiatives in the US, UK and Japan.
A number of key questions must be addressed in the coming decade to harness the potential of
this research: Firstly, why are misfolded proteins toxic to cells? What are the structural features of
these species and what are their cellular targets? Which properties of aberrant proteins are general
and which are disease-specific? Secondly, how does the cellular protein quality machinery
function normally in neutralizing and removing these toxic forms, and what are the reasons for its
failure to maintain protein homeostasis during aging? And finally, how can the formation of toxic
protein aggregates be prevented or, when already present in cells, how can they be removed? For
example, is it possible to develop drug-like molecules that interfere with aggregate formation or
can we activate the cell’s own defense mechanisms, chaperones and proteases, to do the job? For
example, proof-of-principle experiments have already shown that certain drugs can upregulate
molecular chaperones, thereby preventing the aggregation of huntingtin, the disease protein of
Chorea Huntington 7 (Figure 6). Furthermore, the screening of large compound libraries has led
to the identification of substances that can inhibit the aggregation of disease proteins directly.
Research opportunities, needs and challenges
Clearly, understanding a problem as complex as protein folding and cellular quality control
provides research opportunities in many areas of the life sciences in which the Max Planck
Society has already developed a strong focus. Of particular significance in this context is the
recent inauguration of a new Max Planck Institute for Biology of Aging. The main challenge lies
in bringing the efforts of different disciplines together, resulting in an integrated view from
structural biology, cell biology, neurobiology and systems biology. Already ongoing
collaborative activities between different Max Planck Institutes will be expanded to form a virtual
research institute for protein folding, misfolding and disease. Structural analysis by solid-state
NMR and other biophysical methods, coupled with molecular dynamics simulations in silico, will
be employed to determine the molecular features of aggregation intermediates and aggregation
end-states. These investigations will be correlated with toxicity studied in model organisms,
including cells in culture, worm, fly and mouse models 8. Quantitative proteomics will be
employed to identify the cellular targets and interaction partners of misfolded protein species to
provide insight into the mechanism underlying proteotoxicity. Research on the cellular defense
pathways against protein misfolding will focus on a systems approach to define the components
of protein homeostasis and how they cooperate in networks. These efforts will determine how the
structure of these networks changes during aging and identify possible drug targets for the
modulation of molecular chaperones and their regulators. Finally, methods from chemical biology
coupled with the screening of large compound libraries will be used to discover drug-like
molecules that activate the chaperone response in aging cells or interfere directly with misfolding
and aggregation of specific proteins 9. The newly-founded Lead Discovery Center of Max Planck
Innovation in Dortmund will serve in the preclinical evaluation of these molecules.
Expected outcome and benefit
Aberrant protein folding is not only central to understanding some of the most debilitating
diseases of our time but is also fundamental in explaining why we age. Basic research in this area,
conducted with an open mind for possible applications, ultimately has the potential to improve
healthy life span, thereby making an important contribution to a positive social and cultural
development of society. An interaction between natural science disciplines and the humanities
within and beyond the Max Planck Society will be desirable to accompany this process.
Figures
Figure 1A
Structure of a folded protein from X-ray crystallographic analysis. Shown as an example for the
intricacy of the three-dimensional fold is the sugar binding maltose binding protein.
Figure 1B
During folding the linear chain of amino acids (squares, triangles, circles) undergoes an enormous
compaction. The linear chain and the folded structure of MBP are drawn to scale. There are about
1030 possible ways to fold a protein chain like MBP, but only one corresponds to the biologically
active protein.
Figure 2
Aggregation as a side reaction of protein folding. Unfolded and partially folded protein chains
(compact intermediates) expose hydrophobic amino acid residues that can give rise to
aggregation. Molecular chaperones transiently shield these regions, thereby preventing
aggregation. Cycles of chaperone binding and release results in productive folding.
Figure 3
Pathway of chaperone-assisted protein folding in the bacterial cytosol. Trigger factor (TF) binds
directly to the large ribosomal subunit and is the first chaperone to interact with newlysynthesized protein chains. Most small proteins (65-80%) may only interact with TF, but longer
proteins (10-20%) interact subsequently with the Hsp70 system DnaK and DnaJ. Both TF and
Hsp70 recognize short hydrophobic segments in unfolded protein chains that can give rise to
aggregation. Particularly aggregation-prone proteins (10-15% of total) must be transferred into
the central cavity of the cylindrical GroEL chaperonin and fold in isolation upon encapsulation by
the lid-shaped GroES.
Figure 4A
Cross sections of normal and Alzheimer’s disease (AD) brain showing the dramatic atrophy in
regions responsible for memory and language skills. Source: Internet.
Figure 4B
Microscopic image of brain tissue form an Alzheimer disease (AD) patient showing the typical
AD deposits called plaques and tangles. The plaques contain the 42 amino acid long AD peptide
(A beta) and the tangles contain aggregates of the protein Tau and other associated components.
These aggregates are thought to severely disturb the function of neuronal cells. Source: Eckhard
Mandelkow, Max Planck research group, Hamburg.
Figure 5
Structure of amyloid fibrils that are deposited within neuronal cells in Parkinson’s disease. They
consist of the protein alpha-synuclein. The gray background shows the electron microscopic
image from which the threedimensional reconstruction in gold is derived. Source: Christian
Griesinger and Markus Zweckstetter, MPI fuer biophysikalische Chemie, Göttingen.
Figure 6
Activation of molecular chaperones by the drug geldanamycin (GA) prevents aggregation of the
disease protein of Huntington’s disease (huntingtin) in mammalian cells in culture. Huntingtin is
shown as a green fluorescent protein. The control panel shows untreated cells containing large
huntingtin aggregates and the cell nucleus in blue.
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intervention. Science 319, 916-919 (2008).
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