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MECHANISMS
OF IN VIVO PROTEIN FOLDING
In contrast to in vitro refolding experiments,
folding in the cell must be accomplished in the context of the
vectorial synthesis of polypeptide chains on ribosomes. In principle,
the N-terminal portion of the polypeptide could fold spontaneously
as it emerges from the ribosome. On the other hand, the cooperative
nature of the interactions that stabilize folded structures
requires a complete folding domain (100-200 amino acids) to
be available for productive folding. Given the high density
of folding protein molecules emerging from polysomes, this would
imply that the growing polypeptide has to be effectively prevented
from misfolding and aggregating, until a chain length suitable
for productive folding has been synthesized. The present project
proposes to combines biochemical and cell biological approaches
to investigate the folding of newly synthesized proteins. We
have developed two experimental approaches that will yield complementary
information. The first approach relies on the biochemical and
biophysical analysis of newly synthesized proteins using in
cell-free translation lysates derived from eukaryotic cells.
The second approach examines the interaction of molecular chaperones
with newly translated polypeptides in the cytosol of intact
mammalian cells. Using the cell-free translation system we have
uncovered substantial differences between the mechanisms of
chaperone-mediated folding of denatured and newly translated
proteins . These biochemical experiments indicated that the
successful folding of newly-translated proteins is mediated
by a highly organized chaperone machinery. For instance, the
pathway that folds firefly luciferase combines two distinct
principles of chaperone action, namely the Hsp70/Hsp40 proteins,
that act by stabilizing extended polypeptides, and the chaperonin
TRiC, which by virtue of its ring-like structure creates an
environment that is favorable for polypeptide folding. Furthermore,
our in vivo experiments have demonstrated that the cellular
chaperone machinery is coupled to translation to create a protected
environment where newly synthesized proteins fold.
The association of chaperones with nascent polypeptides
raises many questions concerning the biochemistry and cell biology
of protein folding. The goals of our research are:
-To understand the mechanism of chaperone-mediated
folding during translation.
-To define which subsets of cellular proteins
require chaperone assistance to fold in vivo.
-To uncover novel components of the cellular
chaperone machinery and examine the regulation of protein folding
in mammalian cells.
To this end, we are currently developing new
strategies to study the interactions between newly translated
polypeptides and components of the folding machinery. In order
to gain mechanistic insights we are using biochemical and biophysical
approaches to dissect folding following translation in cell-free
systems. On the other hand, we are studying the process of protein
folding in intact mammalian cells. By following the folding
kinetics and chaperone interactions of newly translated polypeptides
in vivo we expect to understand how this complex cellular machinery
determines the fate of proteins in the cell.
1) Biochemical Analysis of Polypeptide Folding
During Translation.
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To study mechanistic and
regulatory aspects of folding during translation, we are
exploiting the in vitro translation system we previously
developed in combination with molecular tools and reagents
that arise from previous biophysical, structural and genetic
studies. A particularly advantageous approach is the incorporation
of photoactivatable crosslinkers and fluorescent probes
into the nascent chains by addition of chemically modified
aminoacyl-tRNAs to the translation reactions . In this method,
lysyl-tRNA's are modified by the addition of a photoactivatable
azido crosslinker moiety in the epsilon-amino group (N3-Lys-tRNA)
(Figure 1 A).
The presence of photoactivatable crosslinkers
allows the identification of novel proteins that bind
to nascent chains (Figure 1 B), while the incorporation
of fluorescent probes facilitates the analysis of folding
intermediates produced during translation. This system
constitutes a powerful tool for the biochemical dissection
of the folding machinery, particularly when combined with
other reagents such as temperature-sensitive chaperone
mutants and truncated mRNAs that generate nascent chains
of defined length. Since this methodology can also be
used to incorporate fluorescent probes into nascent chains,
it provides the means to analyze the folding intermediates
that are generated during translation, including the formation
of folded sub-structures and the effect of binding to
chaperones. We are currently exploring the potential of
this methodology using as model substrates the proteins
actin and luciferase. We have detected crosslinking of
luciferase and actin nascent chains to several chaperone
proteins in the lysate including Hsc70 and TRiC (Figure
1 C for Hsc70).
Using these biochemical approaches we
expect to identify the necessary components that mediate
folding and recruit molecular chaperones to the nascent
chains. For example, by using the binding of the nascent
chain to a specific chaperone as an assay, it will be
possible to identify the factors necessary to recruit
chaperones into a complex that can fold nascent chains.
This approach may identify additional components of the
cellular folding machinery, including factors that catalyze
the covalent and non-covalent modification of newly-synthesized
proteins, such as folding catalysts and enzymes for the
attachment of prosthetic groups. Our ultimate goal will
be the in vitro reconstitution of the translation-folding
machinery using purified components.
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Protein Folding in the cell. |
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As stated above, the main
goal of our studies is to understand how proteins fold in
the cell.
A very important question concerns the
overall contribution of chaperones to folding in vivo
. In principle, it is conceivable that distinct chaperone
pathways exist to regulate the diverse fates of polypeptides
in the cytosol. To investigate this problem we have developed
an experimental system using cultured mammalian cells
capable of inducibly expressing dominant negative chaperone-mutants.
Recently, we have used this system to investigate the
processivity of cellular folding. To this end, we overexpressed
in mammalian cells a bacterial chaperonin "trap",
that irreversibly captures unfolded polypeptides. This
"trap" did not interrupt the productive folding
pathway and was unable to bind newly translated polypeptides
which bound instead to endogenous chaperones. This study
indicated that folding in mammalian cells occurs without
the release of non-native folding intermediates into the
bulk cytosol, and established that de novo protein folding
occurs in a protected environment created by a highly
processive chaperone machinery. It will be of great importance
to determine how the processivity is established. One
contributing factor is probably the coupling of the folding
machinery to translation. To gain understanding into this
question we are planning to identify the cellular factors
that bind to ribosome-bound nascent chains.
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The study of folding in mammalian cells
has also allowed us to address a number of questions regarding
the most characterized cytosolic chaperone system, namely
Hsc70 and TRiC. For instance, using intact mammalian cells
we determined the contribution of the major chaperone
systems in the cytosol to de novo folding. To this end
we examined what fraction of newly translated cellular
proteins transit through these chaperones. Our approach
involves pulse-chase protocols followed by the stabilization
and analysis of the complexes between folding substrates
and endogenous chaperones. We found that a large fraction
of newly translated polypeptides associate transiently
with the molecular chaperones Hsc70 and the chaperonin
TRiC/CCT during their biogenesis (Figure 2). The substrate
repertoire observed for Hsc70 and TRiC is not identical:
Hsc70 interacts with a wide spectrum of polypeptides larger
than 20 kDa, while TRiC associates with a diverse set
of proteins between 30-60 kDa. We observed that approximately
15-20% of the total labeled proteins bound transiently
to Hsc70 and ~9-15% associated with TRiC. The identification
of endogenous chaperone-substrates will allow us to define
the structural features that underlie the requirement
for a specific chaperone system in the cell. Our experiments
have also indicated that a significant portion of newly
synthesized polypeptides (~65%) appear to reach the native
state without the assistance of Hsc70 or TRiC (Figure
2). It is conceivable that a fraction of these polypeptides
folds in a chaperone-independent manner. However, novel
uncharacterized chaperone systems may be responsible for
folding specific subsets of cytosolic proteins. We hope
to identify these novel systems by combining these in
vivo studies with biochemical approaches.
Developing these experimental systems
will allow us to investigate other aspects of protein
folding. The basic chaperone machinery may be used to
assemble more complex macromolecular structures, such
as amyloid deposits and viral particles
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